Detection of point mutations in K- ras gene at codon 12 in bile from percutaneous transhepatic choledochal drainage tubes for diagnosis of biliary strictures

Summary Detection of K-ras mutations at codon 12 constitutes one modality for diagnosis of pancreatic tumors. We attempted to detect K-ras mutations in DNA from bile collected through percutaneous transhepatic choledochal drainage (PTCD) tubes as a diagnostic approach to biliary strictures. Since bile salts induce cell damage, we first investigated the degeneration of cells according to bile exposure time using cell lines. High-mol-wt DNA could be extracted from cells exposed to bile for 6 h, but not from those exposed for 12 h. However, DNA exposed to bile for up 12 h could be amplified by the polymerase chain reaction (PCR) method. Therefore, K-ras mutations in fresh bile specimens collected from 15 patients through PTCD tubes were examined using PCR with restriction enzyme digestion. K-ras mutations were found in five out of five (100%) pancreatic cancers, all of which were negative according to cytodiagnosis of the same bile. On the other hand, K-ras mutations were not detected in bile from biliary tract cancers or metastatic neoplasms, except for one bile duct carcinoma and one metastatic case. Thus, although K-ras mutation alone is not an absolute marker for cancer, detection of K-ras mutations in fresh bile from PTCD tubes is a useful adjunct for diagnosis of pancreatic carcinomas in cases of biliary tract strictures.     

Meconium exposure dependent cell death and apoptosis in human alveolar epithelial cells

Alveolar epithelial cells of neonates are directly exposed to aspirated meconium during meconium aspiration syndrome (MAS). This study was designed to investigate the influence of quantity and time of meconium exposure on the cell viability and caspase activity in type II human alveolar epithelial cells. Human alveolar epithelial cells were incubated with human meconium suspension at different concentrations and for different times. Cell viability and DNA fragmentation were investigated together with caspases activity and the amount of Bcl‐2 protein present. We found that cell viability was significantly lower in cells exposed to a higher concentration of meconium. This was also true for cells exposed to meconium for longer. Significantly higher DNA fragmentation, an approximately two‐ to fivefold increase, was observed in cells that had been exposed to higher (5% and 10%) concentration of meconium compared to those treated with lower (0.1% and 1%) concentrations (P < 0.05). The activity of most apoptotic initiators (caspase 2, 8, 9, 10) and effectors (caspase 3, 6) were found to be significantly higher in cells subject to greater meconium exposure compared to cells with no or minor meconium exposure. The level of Bcl‐2 was also found to be significantly decreased in meconium‐exposed cells (P < 0.05). In conclusion, human meconium would seem to induce direct cell death as well as caspase‐dependent apoptosis in alveolar epithelial cells; the amount and period of exposure to meconium are crucial factors in this process. Thus, removing aspirated meconium should alleviate lung cell damage in neonates and improve the outcome with MAS. Pediatr. Pulmonol. 2010; 45:816–823. © 2010 Wiley‐Liss, Inc.     

Characteristics of death of neonatal rat cardiomyocytes following hypoxia or hypoxia-reoxygenation: the association of apoptosis and cell membrane disintegrity

Irreversibly injured cardiomyocytes are positive for terminal deoxynucleotidyl transferase nick end-labeling (TUNEL), making it controversial as to whether TUNEL-positive cardiomyocytes induced by hypoxia–reoxygenation are apoptotic (secondarily necrotic) or primarily necrotic. We investigated the relationship between plasma membrane integrity and DNA fragmentation in hypoxic-reoxygenated cardiomyocytes. Cardiomyocytes were prepared from neonatal rat heart and exposed to hypoxia. The plasma membrane integrity was assessed by propidium iodide (PI) staining. The mode of DNA fragmentation was assessed by TUNEL and in situ polymerase chain reaction ligation assay. Furthermore, caspase-3 activity was measured in hypoxic-reoxygenated cardiomyocytes. Reoxygenation for 24 h after 3–8 h of hypoxia increased TUNEL positivity. However, the appearance of PI-positivity preceded that of TUNEL at various time points following reoxygenation. In contrast, TUNEL-positive but PI-negative cells were rarely found. In the hypoxic-reoxygenated cells, caspase-3 activity was increased, and PI- and TUNEL-positive cardiomyocytes possessed a sufficient number of double-strand DNA breaks with single-base 3′-OH terminals. In cardiomyocytes subjected to hypoxia–reoxygenation, the appearance of TUNEL positivity was delayed in comparison to membrane disintegrity, but in these cells caspase-3 has been activated and the mode of DNA fragmentation was apoptosis-specific. Thus, hypoxia–reoxygenation induces apoptosis associated with cell membrane disintegrity in cardiomyocytes. Received: December 13, 2001 / Accepted: May 8, 2002 Correspondence to Y. Maruyama     

Dose-dependent preferential binding of polycyclic aromatic hydrocarbons to reiterated DNA of murine skin cells in culture.

The distribution of active metabolites of polycyclic aromatic hydrocarbons bound to reiterated or unique regions of murine DNA has been studied by a DNA-DNA renaturation technique. Murine skin cells were exposed to different doses of radioactive polycyclic aromatic hydrocarbons for 24 hr; then the hydrocarbon-labeled DNA was isolated, fragmented, and denatured. Renaturation kinetics and thermal stabilities of DNA-DNA duplexes were studied. At high carcinogen doses, polycyclic aromatic hydrocarbon adducts seem to be distributed equally among the DNA of all reiteration frequencies. At low carcinogen doses, however, a dose-dependent preferential binding to reiterated DNA sequences occurs. An inverse linear relationship appears to exist between the enrichment of hydrocarbon adducts in reiterated DNA sequences and the logarithm of the amount of total hydrocarbon bound to DNA.     

An early decrease in serum HBeAg titre is a strong predictor of virological response to entecavir in HBeAg-positive patients

Summary. Quantification of HBeAg levels has been found to be useful in monitoring and predicting the outcomes of interferon and lamivudine treatment in HBeAg‐positive patients. The aim of this study was to determine whether quantification of HBeAg at baseline and on treatment could predict which patients would achieve HBeAg seroconversion after 96 weeks of entecavir therapy. Sixty‐five HBeAg‐positive naïve chronic hepatitis B patients who were treated with entecavir at a dose of 0.5 mg once daily for 96 weeks were evaluated. Serum HBV DNA levels were assessed at baseline, week 24, 48 and 96; serum HBeAg levels were assessed at baseline, week 12, 24, 48, 72 and 96. Serum HBeAg levels were associated with a higher likelihood of HBeAg seroconversion to entecavir at weeks 96 than serum HBV DNA levels both at baseline and on treatment (at baseline: OR = 9.932, P = 0.003 vs OR = 5.045, P = 0.036; on treatment: OR = 112.5, P < 0.0001 vs OR = 47.782, P < 0.0001). A maintained reduction in HBeAg > 65% of pretreatment HBeAg values after 24 weeks of entecavir therapy is the strongest predictor for HBeAg seroconversion at week 96 (OR = 70.578, P < 0.0001). Quantification of HBeAg at the start and early during therapy showed a higher predictive value than that of HBV DNA for HBeAg seroconversion by entecavir. A significant decrease in serum HBeAg levels at week 24 may be a useful on‐treatment measurement in the early phase for predicting HBeAg seroconversion and identifying patients who will most likely benefit from finite entecavir treatment.     

Islet amyloid polypeptide promotes beta-cell proliferation in neonatal rat pancreatic islets

Aims/hypothesis: We aimed to clarify the role of islet amyloid polypeptide, which is expressed at early embryonic onset, in the proliferation and cell death of neonatal islet cells. Methods: Fetal islets were prepared from pregnant rats on gestational day 21. Islets were cultured in RPMI 1640 (11.1 mmol/l glucose) + 10 % fetal calf serum (FCS) for 48 h, followed by a 24-h culture period in RPMI 1640 (5.6 mmol/l glucose) + 1 % FCS. The islets were then exposed to rat islet amyloid polypeptide (1–10 nmol/l) for 24 h. Results: Iselt amyloid polypeptide increased islet DNA synthesis (dpm/μg of DNA · 6 h) (control 1 % FCS: 3634 ± 662; 1 nmol/l 6347 ± 1535; 10 nmol/l 5157 ± 769; p < 0.05 islet amyloid polypeptide vs control). In accordance with this, a doubling of the autoradiographic labelling index was seen in immunocytochemically stained islet beta cells after exposure to 1 and 10 nmol/l islet amyloid polypeptide. Islet amyloid polypeptide at 1 nmol/l increased the islet insulin content (202 ± 25 % of control; p < 0.01) and the 24-h medium insulin concentration (1 nmol/l islet amyloid polypeptide: 143 ± 19 % of control; p < 0.05) but at 10 nmol/l islet amyloid polypeptide these changes did not attain statistical difference. Islet amyloid polypeptide did not have any marked effect on the islet cell death frequency, suggesting that islet amyloid polypeptide is a more potent promoter of proliferation than of programmed cell death. Conclusion/interpretation: Our data indicate islet amyloid polypeptide is a potential regulator of proliferation in neonatal pancreatic islet cells, an effect which can partly be attributed to the proliferation of beta cells. [Diabetologia (2001) 44: 1015–1018] Received: 14 March 2001 and in revised form: 7 May 2001     

Effects of X-irradiation in early ontogenesis on the longevity and amount of the S1 nuclease-sensitive DNA sites in adult Drosophila melanogaster

The long-term consequences of the X-irradiationof Drosophila melanogaster fruit flyone-hour eggs with doses of 0.25, 0.50, 0.75,1, 2 and 4 Gy were investigated. Longevityhormesis was observed in males exposed to 0.5Gy and 0.75 Gy, but no longevity increase wasobserved in females. The electrophoreticanalysis has shown that the amount of the DNAsegments resulting from cleavage in S1nuclease-sensitive sites (<3 kb) reached39.2% of the total DNA from control males. DNAfrom the irradiated males had a smaller amountof such fragments (10–30% in differentexperimental groups). These findings indicatethat the longevity hormesis may be associatedwith irradiation-induced long-term structuraland/or functional DNA modifications.     

内皮细胞脂质过氧化损伤增强主动脉平滑肌细胞表达细胞周期素D

为探讨细胞周期素Ｄ在主动脉平滑肌细胞中的表达规律，揭示动脉粥样硬化中平滑肌细胞的增殖机制，在培养在兔主动脉平滑肌细胞中分别加入了同程度的内皮细胞损伤后的条件培养在２４ｈ分别收集各组的平滑肌细胞，进行免疫组织化学染色和细胞核的增殖试验，结果发现，主动脉平滑肌细胞可表达细胞周期素Ｄ，其免疫反应产物主要位地细胞核内，内皮细胞损伤后的条件的培养基能量产滑有细胞表达细胞促进平滑肌细胞的增殖，以上结果提示主动     

High-throughput assays for detection of the F1534C mutation in the voltage-gated sodium channel gene in permethrin-resistant Aedes aegypti and the distribution of this mutation throughout Thailand

Objectives To develop rapid monitoring tools to detect the F1534C permethrin‐resistance mutation in domain IIIS6 of the Aedes aegypti voltage‐gated sodium channel gene and determine the frequency and distribution of this mutation in Thailand. Methods A TaqMan SNP genotyping and an allele specific PCR (AS‐PCR) assay were developed and validated by comparison with DNA sequencing of homozygous susceptible and homozygous resistant laboratory strains, their reciprocal‐cross progenies, and field‐caught mosquitoes. To determine the resistance phenotype of wild‐caught A. aegypti, mosquitoes were exposed to 0.75% permethrin paper. The AS‐PCR assay was used to screen 619 individuals from 20 localities throughout Thailand. Results Overall, both assays gave results consistent with DNA sequencing for laboratory strains of known genotype and for wild‐caught A. aegypti. The only slight discrepancy was for the AS‐PCR method, which overestimated the mutant allele frequency by 1.8% in wild‐caught samples. AS‐PCR assays of permethrin‐exposed samples show that the mutant C1534 allele is very closely associated with the resistant phenotype. However, 19 permethrin‐resistant individuals were homozygous for the wild‐type F1534 allele. DNA sequencing revealed all these individuals were homozygous for two other mutations in domain II, V1016G and S989P, which are known to confer resistance ( Srisawat et al. 2010 ). The F1534C mutation is widespread in Thailand with mutant allele frequencies varying among populations from 0.20 to 1.00. Conclusions These assays can be used for the rapid detection of the F1534C resistance mutation in A. aegypti populations. The F1534C, and other, mutations underlie an extremely high prevalence of pyrethroid resistance in Thailand.     

Messenger RNA levels of lung extracellular matrix proteins during ozone exposure

Continuous exposure of rats to ozone has been shown to result in lung epithelial damage, inflammation, and subsequent increases in collagen content. The main goal of this study was to identify the earliest time point of altered extracellular matrix protein gene expression by utilizing Northern blot analyses of rat lungs continuously exposed to 1.0 ppm ozone for 14 days. An early increase of steady-state fibronectin mRNA levels was observed at 2 days of exposure, prior to the time point of increased type I collagen mRNA, which was seen at 4 days. This increased level of type I collagen mRNA preceded measurable changes in total lung collagen content, observed at 7 days. In addition, peak levels of the growth-related proto-oncogene c-myc mRNA could be correlated with maximal increases of lung DNA content, although the initial increase in c-myc mRNA preceded measurable changes of total lung DNA. The use of specific cDNA probes for measuring altered gene expression can be useful for defining the early cellular and molecular events in ozone-induced lung injury. Offprint requests to: D. J. P. Bassett     

Airflow obstruction in nonsmoking,asbestos-and mixed dust-exposed workers

Obstructive changes in small airways have been described in patients exposed to asbestos and other mineral dusts. The physiologic significance of these small airways abnormalities and their relationship to dust burden and alveolitis remain unclear. We performed bronchoalveolar lavage (BAL) in 30 nonsmoking and 30 age-matched smoking subjects, all with mild asbestos and mixed dust exposure, to determine if parameters of lung dust burden correlated with spirometric evidence of airflow obstruction. Seventeen of 30 nonsmoking subjects and 24 of 30 smoking subjects met spirometric criteria for airflow obstruction. There were significantly more obstructed subjects in both dust exposed groups (P < 0.05) than in an age-matched nondust exposed group. There was, however, no significant difference in the number of obstructed subjects between the smoking and nonsmoking groups. There was no correlation in either group between airflow obstruction and total or differential cell counts, ferruginous bodies, total asbestos fibers, or the percent of free silica in the particulate fraction recovered by BAL. We conclude that evidence of small airways obstruction occurs commonly in occupationally dust exposed subjects and appears to be related to dust exposure per se and not to alveolar inflammation or fiber retention, important factors in the development of alveolitis and interstitial lung disease. Offprint requests to: D. E. Griffith.     

A novel stop codon mutation within the hepatitis B surface gene is detected in the liver but not in the peripheral blood mononuclear cells of HIV‐infected individuals with occult HBV infection

Summary. To characterize occult HBV infection (OHB) in different compartments of HIV+ individuals. This retrospective study involved 38 consecutive HIV+ patients; 24 HBsAg negative (HBV?) and 14 HBsAg positive (HBV+). OHB was assessed in serum samples, liver tissue (LT) and peripheral blood mononuclear cells (PBMC) by genomic amplification of the partial S, X and precore/core regions. HBV genomic analysis was inferred by direct sequencing of PCR products. The intracellular HBV‐DNA was measured by a quantitative real‐time PCR. HBV+ patients were used as a control for HBV replication and genomic profile. In HBV? patients, HBV‐DNA was undetectable in all serum samples, while it was found positive in 7/24 (29%) LT in which genotype D prevailed (57%). HBV‐DNA was found in 6/7 (86%) PBMC of occult‐positive and none of occult‐negative LT. Significantly lower HBV‐DNA load was present in both compartments in OHB+ with respect to the HBV+ group (LT: P = 0.002; PBMC: P = 0.026). In the occult‐positive cases, HBV replication was significantly higher in LT than in PBMC (P = 0.028). A hyper‐mutated S gene in PBMC and a nucleotide mutation at position C695 in LT that produces a translational stop codon at amino acid 181 of the HBs gene characterized OHB. In this group of HIV+ persons, OHB is frequent and exhibits lower replication levels than chronic HBV in the different compartments examined. HBV‐DNA detection in PBMC may offer a useful tool to identify OHB in serum‐negative cases. The novel HBs gene stop codon found in LT could be responsible for reduced production leading to undetectability of HBsAg.     

Enhanced Neutrophil Apoptosis in Neutropenic Patients with Hepatosplenic Schistosomiasis: Evidence of Serum Fas Ligand

Enhanced neutrophil apoptosis has been reported in neutropenic hepatosplenic schistosomiasis. The shortening of neutrophil survival via apoptosis may explain the neutropenia that occur in these patients. However, the regulation of neutrophil apoptosis in hepatosplenic schistosomiasis has not been clearly defined. Neutrophils harvested from neutropenic patients with hepatosplenic (HS) schistosomiasis, (n=25), non-neutropenic patients with hepatointestinal (HI) schistosomiasis (n=10), and age-/gender-matched healthy control subjects (n=10) were incubated with autologous serum. Neutrophils apoptosis was quantified by flow cytometry through determination of propidium iodide nuclear staining and confirmed by DNA gel electrophoresis at 0 (i.e. fresh neutrophils), 4 and 24 h culture. Neutrophils from healthy subjects were also incubated with either 10% heterologous normal or neutropenic serum, with and without anti-Fas ligand antibody. Fas expression was assessed in fresh neutrophils using flow cytometry. Compared with normal healthy neutrophils, and HI neutrophils, neutropenic neutrophils demonstrated greater apoptosis in the presence of autologous serum (P<0.01, 0.05, respectively). Furthermore, compared with normal neutrophils exposed to heterologous normal serum, those exposed to heterologous neutropenic serum exhibited higher apoptosis rates ( P<0.01). Moreover, anti-Fas L antibody attenuated the neutropenic serum-induced neutrophil apoptosis in normal neutrophils. Fas expression was significantly higher in the neutropenic group when compared to both HI and normal healthy controls (P<0.05). In addition, Fas expression by neutrophils was paralleled by high neutrophil apoptosis. On the other hand, neutrophil apoptosis was not correlated to the size of spleen in neutropenic group.

In conclusion, the rate of neutrophil apoptosis is accelerated in patients with neutropenic hepatosplenic schistosomiasisis. These findings suggest that the enhanced neutrophil apoptosis that occurs in neutropenic HS patients is triggered by a serum factor, which is mostly a Fas ligand.     

A mild stress due to hypergravity exposure at youngage increases longevity in Drosophila melanogaster males

Drosophila melanogaster flies were exposed to hypergravity starting at two days of age, the range of gravity levels used being 2.58–7.38 g. No longevity change was observed for exposures of less than 14 days. The longevity of males increased if they were submitted to hypergravity for durations ranging from 14 to 24 days. This increase in longevity was never observed in females. The positive effect of exposure to hypergravity has been replicated in two laboratories using two wild-type strains and different rearing conditions. A short hypergravity exposure seems to be a mild stress, yielding positive effects on longevity. This is in accordance with two previous studies showing a slight longevity increase after heat shock in the nematode Caenorhabditis elegans and in Drosophila melanogaster.     

Clinical course of 161 untreated and tenofovir‐treated chronic hepatitis B pregnant patients in a low hepatitis B virus endemic region

Hepatitis B immunoprophylaxis failure is linked to high maternal viraemia. There is limited North American data on hepatitis B outcomes in pregnancy. Pregnant hepatitis B carriers were enrolled January 2011–December 2014 and offered tenofovir in the 3rd trimester if hepatitis B virus (HBV)‐DNA was >7‐log IU/mL. Outcomes were determined in treated vs untreated patients. In total, 161 women with 169 pregnancies (one twin, 170 infants; median age 32 years), 18% (29/161) HBeAg+ and median HBV‐DNA 2.51 log IU/mL (IQR 1.66–3.65; range 0.8–8.1) were studied. 14.3% (23/161) received tenofovir due to high viral load (16/23, median 74 days, IQR 59–110) or due to liver disease (7/23). In 10/16 treated due to high viraemia, with confirmed adherence, follow‐up HBV‐DNA showed a 5.49 log decline (P = 0.003). In treatment naïve mothers, median alanine aminotransferase (ALT) increased from 17 IU/L (IQR 12–24) to 29 (IQR 18–36) post‐partum (P = 1.5e‐7). In seven highly viraemic mothers who declined therapy (HBV‐DNA >8‐log IU/mL); median ALT increased ~3X from baseline (P < 0.01). 26% (44/169) had Caesarean section with no difference in treated vs untreated subjects. No tenofovir‐treated mothers had renal dysfunction. Data were available on 167/170 infants; in 50.8% (85/167) who completed immunoprophylaxis, 98.8% (84/85, including 12 exposed to tenofovir in utero) were HBV immune. One infant born to an HBeAg+ mother with HBV‐DNA >8‐log IU/mL failed immunoprophylaxis. In this prospective Canadian cohort study, most untreated mothers experienced mild HBV flares. Tenofovir in pregnancy is well tolerated and reduces viral load prior to parturition.     

DNA adducts in human placenta in relation to tobacco smoke exposure and plasma antioxidant status

The DNA adduct 8-hydroxy-2-deoxyguanosine (8-OHdG) has been widely used as a biomarker for oxidative stress. Bulky DNA adducts, which are detectable by the³²P-postlabelling method, provide evidence for exposure to and metabolic activation of large, mainly apolar compounds, e.g. polycyclic aromatic hydrocarbons. We determined both types of adducts in placental tissues of 30 term pregnancies and related the adduct levels to the exposure to tobacco smoke and the plasma antioxidant status. Urine and plasma cotinine concentrations were used to select 10 nonsmokers, 9 nonsmokers exposed to environmental tobacco smoke (ETS) and 11 smoking women. Placental levels of 8-OHdG were 0.84±0.11, 0.90±0.21 and 0.83±0.20/10⁵ deoxyguanosine bases (dG) for nonsmokers, nonsmokers exposed to ETS and smokers, respectively. The differences between the groups were not significant. Smoking women had significantly lower plasma vitamin C and -carotene concentrations than nonsmoking women or nonsmoking women exposed to environmental tobacco smoke. The 8-OHdG adduct level in placental DNA was inversely correlated with the plasma vitamin E concentration (r=–0.47,P<0.05). There was no association between placental 8-OHdG adducts and vitamin A, C and -carotene in plasma. In total, 15 different adducts could be identified in the 30 placenta samples by the³²P-postlabelling method. There was a strong inter-individual variation in both the number of adducts and adduct intensities. No smoking-related or vitamin-related effects on adduct patterns or intensities were found. Our findings suggests that, within the limits of the methods used, tobacco smoke exposure during pregnancy does not lead to a measurable increase in placental DNA adduct levels and that vitamin E appears to have a protective effect on placental 8-OHdG formation.Abbreviations 8-OHdG 8-hydroxy-2-deoxyguanosine - ETS environmental tobacco smoke     

Hypermethylation of <Emphasis Type="Italic">SFRP2</Emphasis> as a Potential Marker for Stool-Based Detection of Colorectal Cancer and Precancerous Lesions

DNA methylation is a key mechanism of colorectal carcinogenesis. Analysis of aberrantly methylation in stool DNA might provide a novel strategy for noninvasive detection of colorectal cancer (CRC). To explore the feasibility of this approach, we have assessed the methylation status of secreted frizzled-related protein gene 2 (SFRP2) in stool samples from patients with CRC with respect to a series of healthy individuals and patients with benign colorectal diseases, using methylation-specific polymerase chain reaction. Methylated SFRP2 occurs in 94.2%, 52.4%, 37.5%, and 16.7% of patients with CRC, adenomas, hyperplstic polyps, and ulcerative colitis, respectively. Of the 24 normal individuals, only 1 revealed methylated DNA. The pilot study revealed that aberrant methylated SFRP2 could be detected frequently in stools from patients with CRC and precancerous lesions. Methylation testing of fecal DNA may be a simple, promising, and noninvasive screening tool for colorectal neoplasia.     

Melatonin enhances antioxidative enzyme gene expression (CAT,GPx, SOD), prevents their UVR‐induced depletion,and protects against the formation of DNA damage (8‐hydroxy‐2'‐deoxyguanosine) in ex vivo human skin

UV radiation (UVR) induces serious structural and functional alterations in human skin leading to skin aging and carcinogenesis. Reactive oxygen species are key players in UVR‐mediated photodamage and induce the DNA‐base‐oxidized, intermediate 8‐hydroxy‐2'‐deoxyguanosine (8‐OHdG). Herein, we report the protective action of melatonin against UVR‐induced 8‐OHdG formation and depletion of antioxidative enzymes using ex vivo human full‐thickness skin exposed to UVR in a dose (0, 100, 300 mJ/cm²)‐ and time‐dependent manner (0, 24, 48 hr post‐UVR). Dynamics of depletion of antioxidative enzymes including catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD), or 8‐OHdG formation were studied by real‐time PCR and immunofluorescence/immunohistochemical staining. UVR‐treated skin revealed significant and immediate (0 hr 300 mJ/cm²) reduction of gene expression, and this effect intensified within 24 hr post‐UVR. Simultaneous increase in 8‐OHdG‐positive keratinocytes occurred already after 0 hr post‐UVR reaching 71% and 99% up‐regulation at 100 and 300 mJ/cm², respectively (P < 0.001). Preincubation with melatonin (10^?3 m ) led to 32% and 29% significant reductions in 8‐OHdG‐positive cells and the prevention of antioxidative enzyme gene and protein suppression. Thus, melatonin was shown to play a crucial role as a potent antioxidant and DNA protectant against UVR‐induced oxidative damage in human skin.     

The Antiinflammatory Activity of Budesonide on Human Airway Epithelial Cells is Lasting After Removal of the Drug from Cultures

Because of its ability to conjugate extensively with fatty acids within lung cells, it has been suggested that budesonide (Bud) may have a prolonged pharmacologic activity, related to retention of the drug in airway tissues. Using human bronchial epithelial cells (HBECs) as target cells, we evaluated whether Bud could have a long-lasting inhibitory effect on ICAM-1 expression and GM-CSF release. HBECs were cultured in Bud (10 µM) or in medium alone (Ctr) for 24 hr, then extensively washed (to remove Bud) and incubated for an additional 6, 12, or 24 hr with IFN-γ. ICAM-1 expression and GM-CSF release were then measured by flow cytometric analysis. In Ctr HBECs, IFN-γ induced a time-dependent upregulation of ICAM-1 expression, significant at 6, 12, or 24 hr (p < 0.05, each comparison), and an increase in GM-CSF release, significant at 24 hr (p < 0.05). The inhibitory effects of Bud preexposure on IFN-γ-induced ICAM-1 expression and GM-CSF release were then compared with those of a continuous exposure to the drug during IFN-γ stimulation. Preexposure to Bud (1 and 10 µM) induced a significant inhibition of IFN-γ-induced ICAM-1 expression (p < 0.05, each comparison), but lower than that observed in HBECs continuously exposed at the same Bud concentrations (p < 0.01, each comparison). In contrast, the inhibition of GM-CSF release was similar in preexposed and in exposed HBECs and statistically significant only at the highest Bud concentration tested (p < 0.05, each comparison). Thus, Bud is effective in vitro in inducing a downregulation lasting 24 hr of mechanisms involved in leukocyte recruitment.     

Use of recombinant proteins MPB70 or MPB83 as capture antigens in ELISAs to confirm bovine tuberculosis infections in Brazil

The aim of this work was to analyze the content of total protein and nitrogen degradation products in Biomphalaria glabrata infected with Schistosoma mansoni and exposed to Euphorbia splendens var. hislopii latex. The LC₅₀ of this latex was 1.0 mg/l. Concentrations of uric acid, urea and total proteins were determined in the hemolymph of B. glabrata infected with five S. mansoni miracidia and exposed to a sublethal concentration of E. splendens var. hislopii latex for 24 h. The exposure to this molluscicide caused total depletion of the alterative sources of energy (total proteins) and significant variation in the nitrogen degradation products. The urea content increased while the uric acid level decreased. These results reflect a disturbance in the snails regulation of their metabolism due to intoxication caused by the latex exposure.