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Background: A combined modality treatment employing radiation and chemotherapy plays a central role in the management of solid tumors. In our study, we examined the cytotoxic and radiosensitive effect of the microtubule stabilizer epothilone B on two human epithelial tumor cell lines in vitro and its influence on the microtubule assembly.Methods: Cancer cells were treated with epothilone B in proliferation assays and in combination with radiation in colony-forming assays. For the analysis of ionizing radiation-induced DNA damage and the influence of the drug on its repair a γH2AX foci assay was used. To determine the effect of epothilone B on the microtubule assembly in cells and on purified tubulin, immunofluorescence staining and tubulin polymerization assay, respectively, were conducted.Results: Epothilone B induced a concentration- and application-dependent antiproliferative effect on the cells, with IC(50) values in the low nanomolar range. Colony forming assays showed a synergistic radiosensitive effect on both cell lines which was dependent on incubation time and applied concentration of epothilone B. The γH2AX assays demonstrated that ionizing radiation combined with the drug resulted in a concentration-dependent increase in the number of double-strand breaks and suggested a reduction in DNA repair capacity. Epothilone B produced enhanced microtubule bundling and abnormal spindle formation as revealed by immunofluorescence microscopy and caused microtubule formation from purified tubulin.Conclusion: The results of this study showed that epothilone B displays cytotoxic antitumor activity at low nanomolar concentrations and also enhances the radiation response in the tumor cells tested; this may be induced by a reduced DNA repair capacity triggered by epothilone B. It was also demonstrated that epothilone B in fact targets microtubules in a more effective manner than paclitaxel. [ABSTRACT FROM AUTHOR]

Background: Many expert panels recommend colorectal cancer screening for average-risk asymptomatic individuals older than 50 years of age. Recent studies have found that 24% to 64% of primary care providers use only the digital fecal occult blood test (FOBT) as their primary screening test. The effectiveness of a single digital FOBT is unknown.Objective: To compare the sensitivity and specificity of digital FOBT and the recommended 6-sample at-home FOBT for advanced neoplasia in asymptomatic persons.Design: Prospective cohort study.Setting: 13 Veterans Affairs medical centers.Patients: 3121 asymptomatic patients 50 to 75 years of age.Intervention: 2665 patients had 6-sample at-home FOBT and digital FOBT, followed by complete colonoscopy.Measurements: We measured the sensitivity of digital and 6-sample FOBT for advanced neoplasia and the specificity for no neoplasia. We calculated predictive values and likelihood ratios for advanced neoplasia, defined as tubular adenomas 10 mm or greater, adenomas with villous histology or high-grade dysplasia, or invasive cancer.Results: Of all participants, 96.8% were men; their average age was 63.1 years. The 6-sample FOBT and the single digital FOBT had specificities of 93.9% and 97.5%, respectively, as defined by studying 1656 patients with no neoplasia. Sensitivities for detection of advanced neoplasia in 284 patients were 23.9% for the 6-sample FOBT and 4.9% for the digital FOBT. The likelihood ratio for advanced neoplasia was 1.68 (95% CI, 0.96 to 2.94) for positive results on digital FOBT and 0.98 (CI, 0.95 to 1.01) for negative results.Limitations: Most patients were men.Conclusions: Single digital FOBT is a poor screening method for colorectal neoplasia and cannot be recommended as the only test. When digital FOBT is performed as part of a primary care physical examination, negative results do not decrease the odds of advanced neoplasia. Persons with these results should be offered at-home 6-sample FOBT or another type of screening test. [ABSTRACT FROM AUTHOR]

BACKGROUND: This multicenter prospective, randomized, double-blind placebo-controlled trial was designed to evaluate the effectiveness of polyenylphosphatidylcholine against the progression of liver fibrosis toward cirrhosis in alcoholics. Seven hundred eighty-nine alcoholics with an average intake of 16 drinks per day were enrolled. To control excessive drinking, patients were referred to a standard 12-step-based alcoholism treatment program, but most patients refused to attend. Accordingly, study follow-up procedures incorporated the essential features of the brief-intervention approach. An overall substantial and sustained reduction in drinking was observed. Hepatic histological and other findings are described in a companion article. METHODS: Patients were randomized to receive daily three tablets of either polyenylphosphatidylcholine or placebo. Monthly follow-up visits included an extensive session with a medical nurse along with brief visits with a study physician (hepatologist or gastroenterologist). A detailed physical examination occurred every 6 months. In addition, telephone consultations with the nurse were readily available. All patients had a liver biopsy before entry; a repeat biopsy was scheduled at 24 and 48 months. RESULTS: There was a striking decrease in average daily alcohol intake to approximately 2.5 drinks per day. This was sustained over the course of the trial, lasting from 2 to 6 years. The effect was similar both in early dropouts and long-term patients, i.e., those with a 24-month biopsy or beyond. CONCLUSIONS: In a treatment trial of alcoholic liver fibrosis, a striking reduction in alcohol consumption from 16 to 2.5 daily drinks was achieved with a brief-intervention approach, which consisted of a relative economy of therapeutic efforts that relied mainly on treatment sessions with a medical nurse accompanied by shorter reinforcing visits with a physician. This approach deserves generalization to address the heavy drinking problems commonly encountered in primary care and medical specialty practices. [ABSTRACT FROM AUTHOR]

Introduction: Limited data inform the current postpolypectomy surveillance guidelines, which suggest a shortened interval to third colonoscopy after a negative second examination if high-risk adenomas (HRA) were present on the initial screening colonoscopy. Therefore, we examined the risk of HRA at third colonoscopy stratified by findings on 2 previous examinations in a prospective screening colonoscopy cohort of US veterans., Methods: We identified participants who had 3 or more colonoscopies from CSP#380. We examined the risk of HRA on the third examination based on findings from the previous 2 examinations. Multivariate logistic regression was used to adjust for multiple covariates., Results: HRA were found at the third examination in 114 (12.8%) of 891 participants. Those with HRA on both previous examinations had the greatest incidence of HRA at third examination (14/56, 25.0%). Compared with those with no adenomas on both previous examinations, participants with HRA on the first examination remained at significantly increased risk for HRA at the third examination at 3 years after a negative second examination (odds ratio [OR] 3.41, 95% confidence interval [CI] 1.28-9.08), 5 years (OR 3.14, 95% CI 1.49-6.61), and 7 years (OR 2.89, 95% CI 1.08-7.74)., Discussion: In a screening population, HRA on the first examination identified individuals who remained at increased risk for HRA at the third examination, even after a negative second examination. This finding supports current colorectal cancer surveillance guidelines, which suggest a shortened, 5-year time interval to third colonoscopy after a negative second examination if high-risk findings were present on the baseline examination.

Background: Improving the neuronal yield from in vitro cultivated neural progenitor cells (NPCs) is an essential challenge in transplantation therapy in neurological disorders. In this regard, Ascorbic acid (AA) is widely used to expand neurogenesis from NPCs in cultures although the mechanisms of its action remain unclear. Neurogenesis from NPCs is regulated by the redox-sensitive WNT/β-catenin signaling pathway. We therefore aimed to investigate how AA interacts with this pathway and potentiates neurogenesis., Methods: Effects of 200 μM AA were compared with the pro-neurogenic reagent and WNT/β-catenin signaling agonist lithium chloride (LiCl), and molecules with antioxidant activities i.e. N-acetyl-L-cysteine (NAC) and ruthenium red (RuR), in differentiating neural progenitor ReNcell VM cells. Cells were supplemented with reagents for two periods of treatment: a full period encompassing the whole differentiation process versus an early short period that is restricted to the cell fate commitment stage. Intracellular redox balance and reactive oxygen species (ROS) metabolism were examined by flow cytometry using redox and ROS sensors. Confocal microscopy was performed to assess cell viability, neuronal yield, and levels of two proteins: Nucleoredoxin (NXN) and the WNT/β-catenin signaling component Dishevelled 2 (DVL2). TUBB3 and MYC gene responses were evaluated by quantitative real-time PCR. DVL2-NXN complex dissociation was measured by fluorescence resonance energy transfer (FRET)., Results: In contrast to NAC which predictably exhibited an antioxidant effect, AA treatment enhanced ROS metabolism with no cytotoxic induction. Both drugs altered ROS levels only at the early stage of the differentiation as no changes were held beyond the neuronal fate commitment stage. FRET studies showed that AA treatment accelerated the redox-dependent release of the initial pool of DVL2 from its sequestration by NXN, while RuR treatment hampered the dissociation of the two proteins. Accordingly, AA increased WNT/β-catenin signaling output i.e. MYC mRNA level, whereas RuR attenuated it. Moreover, AA improved neurogenesis as much as LiCl as both TUBB3-positive cell yield and TUBB3 mRNA level increased, while NAC or RuR attenuated neurogenesis. Markedly, the neurogenesis outputs between the short and the full treatment with either NAC or AA were found unchanged, supporting our model that neuronal yield is altered by events taking place at the early phase of differentiation., Conclusions: Our findings demonstrate that AA treatment elevates ROS metabolism in a non-lethal manner prior to the NPCs commitment to their neuronal fate. Such effect stimulates the redox-sensitive DVL2 activation and WNT/β-catenin signaling response that would enhance the ensuing neuronal cell differentiation.

BCL-2 is a multifunctional protein involved in the regulation of apoptosis, cell cycle progression and neural developmental processes. Its function in the latter process is not well understood and needs further elucidation. Therefore, we characterized the protein expression kinetics of BCL-2 and associated regulatory proteins of the intrinsic apoptosis pathway during the process of neuronal differentiation in ReNcell VM cells with and without functional inhibition of BCL-2 by its competitive ligand HA14-1. Inhibition of BCL-2 caused a diminished BCL-2 expression and higher levels of cleaved BAX, activated Caspase-3 and cleaved PARP, all pro-apoptotic markers, when compared with untreated differentiating cells. In parallel, flow cytometric analysis of HA14-1-treated cells revealed a delayed differentiation into HuC/D+ neuronal cells when compared to untreated differentiating cells. In conclusion, BCL-2 possess a protective function in fully differentiated ReNcell VM cells. We propose that the pro-survival signaling of BCL-2 is closely connected with its stimulatory effects on neurogenesis of human neural progenitor cells., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

There is an increasing need to develop improved systems for predicting the safety of xenobiotics. However, to move beyond hazard identification the available concentration of the test compounds needs to be incorporated. In this study cyclosporine A (CsA) was used as a model compound to assess the kinetic profiles in two rodent brain cell cultures after single and repeated exposures. CsA induced-cyclophilin B (Cyp-B) secretion was also determined as CsA-specific pharmacodynamic endpoint. Since CsA is a potent p-glycoprotein substrate, the ability of this compound to cross the blood-brain barrier (BBB) was also investigated using an in vitro bovine model with repeated exposures up to 14 days. Finally, CsA uptake mechanisms were studied using a parallel artificial membrane assay (PAMPA) in combination with a Caco-2 model. Kinetic results indicate a low intracellular CsA uptake, with no marked bioaccumulation or biotransformation. In addition, only low CsA amounts crossed the BBB. PAMPA and Caco-2 experiments revealed that CsA is mostly trapped to lipophilic compartments and exits the cell apically via active transport. Thus, although CsA is unlikely to enter the brain at cytotoxic concentrations, it may cause alterations in electrical activity and is likely to increase the CNS concentration of other compounds by occupying the BBBs extrusion capacity. Such an integrated testing system, incorporating BBB, brain culture models and kinetics could be applied for assessing neurotoxicity potential of compounds., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The present study was performed in an attempt to develop an in vitro integrated testing strategy (ITS) to evaluate drug-induced neurotoxicity. A number of endpoints were analyzed using two complementary brain cell culture models and an in vitro blood-brain barrier (BBB) model after single and repeated exposure treatments with selected drugs that covered the major biological, pharmacological and neuro-toxicological responses. Furthermore, four drugs (diazepam, cyclosporine A, chlorpromazine and amiodarone) were tested more in depth as representatives of different classes of neurotoxicants, inducing toxicity through different pathways of toxicity. The developed in vitro BBB model allowed detection of toxic effects at the level of BBB and evaluation of drug transport through the barrier for predicting free brain concentrations of the studied drugs. The measurement of neuronal electrical activity was found to be a sensitive tool to predict the neuroactivity and neurotoxicity of drugs after acute exposure. The histotypic 3D re-aggregating brain cell cultures, containing all brain cell types, were found to be well suited for OMICs analyses after both acute and long term treatment. The obtained data suggest that an in vitro ITS based on the information obtained from BBB studies and combined with metabolomics, proteomics and neuronal electrical activity measurements performed in stable in vitro neuronal cell culture systems, has high potential to improve current in vitro drug-induced neurotoxicity evaluation., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The difficulty in mimicking nervous system complexity and cell-cell interactions as well as the lack of kinetics information has limited the use of in vitro neurotoxicity data. Here, we assessed the biokinetic profile as well as the neurotoxicity of Amiodarone after acute and repeated exposure in two advanced rodent brain cell culture models, consisting of both neurons and glial cells organized in 2 or 3 dimensions to mimic the brain histiotypic structure and function. A strategy was applied to evidence the abiotic processes possibly affecting Amiodarone in vitro bioavailability, showing its ability to adsorb to the plastic devices. At clinically relevant Amiodarone concentrations, known to induce neurotoxicity in some patients during therapeutic treatment, a complete uptake was observed in both models in 24 h, after single exposure. After repeated treatments, bioaccumulation was observed, especially in the 3D cell model, together with a greater alteration of neurotoxicity markers. After 14 days, Amiodarone major oxidative metabolite (mono-N-desethylamiodarone) was detected at limited levels, indicating the presence of active drug metabolism enzymes (i.e. cytochrome P450) in both models. The assessment of biokinetics provides useful information on the relevance of in vitro toxicity data and should be considered in the design of an Integrated Testing Strategy aimed to identify specific neurotoxic alerts, and to improve the neurotoxicity assay predictivity for human acute and repeated exposure., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Apoptosis is an essential physiological process accompanying the development of the central nervous system and human neurogenesis. However, the time scale and the underlying molecular mechanisms are yet poorly understood. Due to this fact, we investigated the functionality and general inducibility of apoptosis in the human neural ReNcell VM progenitor cell line during differentiation and also after exposure to staurosporine (STS) and ultraviolet B (UVB) irradiation. Transmission light microscopy, flow cytometry, and Western-/Immunoblot analysis were performed to compare proliferating and differentiating, in addition to STS- and UVB-treated cells. In particular, from 24 to 72 h post-initiation of differentiation, G0/G1 cell cycle arrest, increased loss of apoptotic cells, activation of pro-apoptotic BAX, Caspase-3, and cleavage of its substrate PARP were observed during cell differentiation and, to a higher extent, after treatment with STS and UVB. We conclude that redundant or defective cells are eliminated by apoptosis, while otherwise fully differentiated cells were less responsive to apoptosis induction by STS than proliferating cells, likely as a result of reduced APAF-1 expression, and increased levels of BCL-2. These data provide the evidence that apoptotic mechanisms in the neural ReNcell VM progenitor cell line are not only functional, but also inducible by external stimuli like growth factor withdrawal or treatment with STS and UVB, which marks this cell line as a suitable model to investigate apoptosis signaling pathways in respect to the differentiation processes of human neural progenitor cells in vitro.

Emerging evidence suggests that reactive oxygen species (ROS) can stimulate the Wnt/β-catenin pathway in a number of cellular processes. However, potential sources of endogenous ROS have not been thoroughly explored. Here, we show that growth factor depletion in human neural progenitor cells induces ROS production in mitochondria. Elevated ROS levels augment activation of Wnt/β-catenin signaling that regulates neural differentiation. We find that growth factor depletion stimulates the release of Ca(2+) from the endoplasmic reticulum stores. Ca(2+) subsequently accumulates in the mitochondria and triggers ROS production. The inhibition of mitochondrial Ca(2+) uptake with simultaneous growth factor depletion prevents the rise in ROS metabolism. Moreover, low ROS levels block the dissociation of the Wnt effector Dishevelled from nucleoredoxin. Attenuation of the response amplitudes of pathway effectors delays the onset of the Wnt/β-catenin pathway activation and results in markedly impaired neuronal differentiation. Our findings reveal Ca(2+)-mediated ROS metabolic cues that fine-tune the efficiency of cell differentiation by modulating the extent of the Wnt/β-catenin signaling output., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Dynamic phase microscopy was used to study the dynamic events of formation of the endoplasmic reticulum (ER) in interphase-arrested Xenopus egg extract. We have shown that the ER periodically oscillated in an ATP-dependent manner in the frequency range of 1.6-2.2 Hz, while the tubular membrane network formed in vitro. The spectral density, i.e. the pattern of a given frequency component in the Fourier spectrum, was strongly correlated with the dynamic events during microtubule-dependent and microtubule-independent ER network formation observed by video-enhanced contrast differential interference contrast and fluorescence microscopy. Because the 1.6-2.2 Hz frequency of oscillation during the network formation was detected both in the presence and absence of microtubules, it appears to be an intrinsic ATP-dependent ER membrane property. Several characteristic active and inactive stages of ER network formation were observed both in the presence and absence of microtubules. However, data analysis of these stages indicated that microtubules and dynein motor activity have a strong influence and a cooperative effect on the kinetics of ER formation by controlled fusion reaction.

It was suggested that gap junctional intercellular communication (GJIC) and connexin (Cx) proteins play a crucial role in cell proliferation and differentiation. However, the mechanisms of cell coupling in regulating cell fate during embryonic development are poorly understood. To study the role of GJIC in proliferation and differentiation, we used a human neural progenitor cell line derived from the ventral mesencephalon. Fluorescence recovery after photobleaching (FRAP) showed that dye coupling was extensive in proliferating cells but diminished after the induction of differentiation, as indicated by a 2.5-fold increase of the half-time of fluorescence recovery. Notably, recovery half-time decreased strongly (five-fold) in the later stage of differentiation. Western blot analysis revealed a similar time-dependent expression profile of Cx43, acting as the main gap junction-forming protein. Interestingly, large amounts of cytoplasmic Cx43 were retained mainly in the Golgi network during proliferation but decreased when differentiation was induced. Furthermore, down-regulation of Cx43 by small interfering RNA reduced functional cell coupling, which in turn resulted in a 50% decrease of both the proliferation rate and neuronal differentiation. Our findings suggest a dual function of Cx43 and GJIC in the neural development of ReNcell VM197 human progenitor cells. GJIC accompanied by high Cx43 expression is necessary (1) to maintain cells in a proliferative state and (2) to complete neuronal differentiation, including the establishment of a neural network. However, uncoupling of cells is crucial in the early stage of differentiation during cell fate commitment., (© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Glycogen synthase kinase-3beta is a multifunctional key regulator enzyme in neural developmental processes and a main component of the canonical Wnt signaling pathway. It is already known that the Wnt-driven differentiation of neural progenitor cells is accompanied by an increase of apoptosis at which the pro-apoptotic function of GSK-3beta is still discussed. The aim of the present study was to investigate whether the phosphorylation level of GSK-3beta at serine 9 is the primary regulatory mechanism of differentiation-induced apoptosis. Differentiating human neural ReNcell VM progenitor cells were treated with the specific GSK-3beta inhibitor SB216763 (10 μM) and analyzed in respect to the intrinsic apoptosis pathway regulation using microscopy and protein expression analysis. Differentiation of ReNcell VM cells was accompanied by cell morphological changes, cytoskeleton rearrangement and apoptosis increase. Treatment of differentiating cells with SB216763 induced a significant dephosphorylation of GSK-3beta at serine 9 accompanied by a significant decrease of apoptosis of about 0.7±0.03% and reduced activation of caspase-3 as well as BAX and PARP cleavage during the first 12h of differentiation compared to untreated, differentiating cells. Dephosphorylation of GSK-3beta at serine 9 appears not solely to be responsible for its pro-apoptotic function, because we observed a decrease of intrinsic apoptosis after treatment of the cells with the specific GSK-3beta inhibitor SB216763. We assume that GSK-3beta drives neural progenitor cell apoptosis by direct interaction with pro-apoptotic BAX or by indirect influence on the canonical Wnt/beta-catenin target gene transcription., (Copyright © 2012 ISDN. Published by Elsevier Ltd. All rights reserved.)

Since nano-sized particles (NPs) are increasingly used in various fields of innovative biomedicine and industrial technologies, it is of importance to identify their potential human health risk. We investigated whether ROS-induced mitochondrial DNA damage is the mode of action of titanium dioxide-NPs (TiO2-NPs; ≤20 nm) to induce cytotoxic and genotoxic effects in human HaCaT keratinocytes in vitro. We showed that TiO2-NPs accumulate at the cell surface and are taken up by endocytosis. Micronucleus (MN) formation was found to be significantly maximal increased 24 h after treatment with 10 μg/ml and 48 h after treatment with 5 μg/ml TiO2-NPs about 1.8-fold respectively 2.2-fold of control. Mitochondrial DNA damage measured as "common deletion" was observed to be significantly 14-fold increased 72 h after treatment with 10 μg/ml TiO2-NPs when compared to control. Four hours after treatment with 5 and 50 μg/ml TiO2-NPs the level of ROS in HaCaT cells was found to be significantly increased about 7.5-fold respectively 16.7-fold of control. In conclusion, for the first time we demonstrate the induction of the mitochondrial "common deletion" in HaCaT cells following exposure to TiO2-NPs, which strongly suggests a ROS-mediated cytotoxic and genotoxic potential of NPs. However, the effects of the modification of TiO2-NPs, such as agglomeration, size distribution pattern and exposure time have to be further critically examined., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

ReNcell VM is an immortalized human neural progenitor cell line with the ability to differentiate in vitro into astrocytes and neurons, in which the Wnt/β-catenin pathway is known to be involved. However, little is known about kinetic changes of this pathway in human neural progenitor cell differentiation. In the present study, we provide a quantitative profile of Wnt/β-catenin pathway dynamics showing its spatio-temporal regulation during ReNcell VM cell differentiation. We show first that T-cell factor dependent transcription can be activated by stabilized β-catenin. Furthermore, endogenous Wnt ligands, pathway receptors and signaling molecules are temporally controlled, demonstrating changes related to differentiation stages. During the first three hours of differentiation the signaling molecules LRP6, Dvl2 and β-catenin are spatio-temporally regulated between distinct cellular compartments. From 24 h onward, components of the Wnt/β-catenin pathway are strongly activated and regulated as shown by mRNA up-regulation of Wnt ligands (Wnt5a and Wnt7a), receptors including Frizzled-2, -3, -6, -7, and -9, and co-receptors, and target genes including Axin2. This detailed temporal profile of the Wnt/β-catenin pathway is a first step to understand, control and to orientate, in vitro, human neural progenitor cell differentiation.

Novel dermorphin tetrapeptides are described in which Tyr(1) is replaced by Dmt(1), where d-Ala(2) and Gly(4) are N-methylated, and where Phe(3)-Gly(4) residue is substituted by the constrained Aba(3)-Gly(4) peptidomimetic. Most of these peptidic ligands displayed binding affinities in the nanomolar range for both μ- and δ-opioid receptors but no detectable affinity for the κ-opioid receptor. Measurements of cAMP accumulation, phosphorylation of extracellular signal-regulated kinase (ERK1/2) in HEK293 cells stably expressing each of these receptors individually, and functional screening in primary neuronal cultures confirmed the potent agonistic properties of these peptides. The most potent ligand H-Dmt-NMe-d-Ala-Aba-Gly-NH(2) (BVD03) displayed mixed μ/δ opioid agonist properties with picomolar functional potencies. Functional electrophysiological in vitro assays using primary cortical and spinal cord networks showed that this analogue possessed electrophysiological similarity toward gabapentin and sufentanil, which makes it an interesting candidate for further study as an analgesic for neuropathic pain.

A series of analogues of conjugate 1, combining an adamantane-based paclitaxel (taxol) mimetic with colchicine was synthesized and tested for cytotoxicity in a cell-based assay with the human lung carcinoma cell line A549. The most active compounds (10 EC(50) 2 ± 1.0 nM, 23 EC(50) 6 ± 1.4 nM, 26 EC(50) 5 ± 1.8 nM, 28 EC(50) 11 ± 1.7 nM, 30 EC(50) 4.8 ± 0.5 nM) were found to interfere with the microtubule dynamics in an interesting manner. Treatment of the cells with these compounds promoted disassembly of microtubules followed by the formation of stable tubulin clusters. Structure-activity relationships for the analogues of 23 revealed the sensitivity of both cytotoxicity and tubulin clustering ability to the linker length. The presence of adamantane (or another bulky hydrophobic and non-aromatic moiety) in 23 was found to play an important role in the formation of tubulin clusters. Structural requirements for optimal activity have been partially explained by molecular modeling., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Because of its diverse physical and chemical properties, lanthanum has been used in various industrial and medical fields. However, until recently, its effects at the cellular and molecular level had hardly been investigated. Using primary cortical networks grown on microelectrode array neurochips, we investigated the acute functional neurotoxicity of lanthanum(III) chloride (LaCl(3)). Lanthanum caused a biphasic concentration-dependent decline in network activity resulting in a complete cessation of the activity at 3mM LaCl(3). However, the networks' oscillatory behavior and synchronicity between neurons remained unaffected until activity loss. The spike activity diminished at half effective concentration values for the two phases of 117 nM and 763 μM LaCl(3) corresponding to 16 ng/ml and 10.6 μg/ml lanthanum, respectively. Furthermore, under the experimental conditions, LaCl(3) did not affect voltage-dependent ion channels contributing to the shape and amplitude of the action potential. Further similarity analysis by pattern recognition exposed significant similarities of the activity changes caused by LaCl(3) to those induced by phenobarbital, gamma-aminobutyric acid, and the gap junction blocker carbenoxolone and sodium propionate. Overall, this study demonstrates inhibitory and potentially sedative toxicological effects of lanthanum(III) ions at concentrations comparable to the plasma concentrations observed in patients with kidney disease being treated with lanthanum carbonate for hyperphosphatemia. Therefore, given the lack of proof that the blood-brain barrier is completely impermeable in uremic patients and lanthanum cannot cross, caution is warranted.

Remodelling of the actin cytoskeleton plays a key role in particle internalisation and the phagosome maturation processes. Actin-binding proteins (ABPs) are the main players in actin remodelling but the precise role of these proteins in phagocytosis needs to be clarified. Annexins, a group of ABPs, are known to be present on phagosomes. Here, we identified annexin A1 as a factor that binds to isolated latex bead phagosomes (LBPs) in the presence of Ca(2+) and facilitates the F-actin-LBP interaction in vitro. In macrophages the association of endogenous annexin A1 with LBP membranes was strongly correlated with the spatial and temporal accumulation of F-actin at the LBP. Annexin A1 was found on phagocytic cups and around early phagosomes, where the F-actin was prominently concentrated. After uptake was completed, annexin A1, along with F-actin, dissociated from the nascent LBP surface. At later stages of phagocytosis annexin A1 transiently concentrated only around those LBPs that showed transient F-actin accumulation ('actin flashing'). Downregulation of annexin A1 expression resulted in impaired phagocytosis and actin flashing. These data identify annexin A1 as an important component of phagocytosis that appears to link actin accumulation to different steps of phagosome formation.

Due to lack of knowledge only a few industrial chemicals have been identified as developmental neurotoxicants. Current developmental neurotoxicity (DNT) guidelines (OECD and EPA) are based entirely on in vivo studies that are both time consuming and costly. Consequently, there is a high demand to develop alternative in vitro methods for initial screening to prioritize chemicals for further DNT testing. One of the most promising tools for neurotoxicity assessment is the measurement of neuronal electrical activity using micro-electrode arrays (MEAs) that provides a functional and neuronal specific endpoint that until now has been used mainly to detect acute neurotoxicity. Here, electrical activity measurements were evaluated to be a suitable endpoint for the detection of potential developmental neurotoxicants. Initially, primary cortical neurons grown on MEA chips were characterized for different cell markers over time, using immunocytochemistry. Our results show that primary cortical neurons could be a promising in vitro model for DNT testing since some of the most critical neurodevelopment processes such as progenitor cell commitment, proliferation and differentiation of astrocytes and maturation of neurons are present. To evaluate if electrical activity could be a suitable endpoint to detect chemicals with DNT effects, our model was exposed to domoic acid (DomA), a potential developmental neurotoxicant for up to 4 weeks. Long-term exposure to a low concentration (50nM) of DomA increased the basal spontaneous electrical activity as measured by spike and burst rates. Moreover, the effect induced by the GABA(A) receptor antagonist bicuculline was significantly lower in the DomA treated cultures than in the untreated ones. The MEA measurements indicate that chronic exposure to DomA changed the spontaneous electrical activity leading to the possible neuronal mal functioning. The obtained results suggest that the MEAs could be a useful tool to identify compounds with DNT potential., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: 3D-scaffolds have been shown to direct cell growth and differentiation in many different cell types, with the formation and functionalisation of the 3D-microenviroment being important in determining the fate of the embedded cells. Here we used a hydrogel-based scaffold to investigate the influences of matrix concentration and functionalisation with laminin on the formation of the scaffolds, and the effect of these scaffolds on human neural progenitor cells cultured within them., Methods: In this study we used different concentrations of the hydrogel-based matrix PuraMatrix. In some experiments we functionalised the matrix with laminin I. The impact of concentration and treatment with laminin on the formation of the scaffold was examined with atomic force microscopy. Cells from a human fetal neural progenitor cell line were cultured in the different matrices, as well as in a 2D culture system, and were subsequently analysed with antibody stainings against neuronal markers. In parallel, the survival rate of the cells was determined by a live/dead assay., Results: Atomic force microscopy measurements demonstrated that the matrices are formed by networks of isolated PuraMatrix fibres and aggregates of fibres. An increase of the hydrogel concentration led to a decrease in the mesh size of the scaffolds and functionalisation with laminin promoted aggregation of the fibres (bundle formation), which further reduces the density of isolated fibres. We showed that laminin-functionalisation is essential for human neural progenitor cells to build up 3D-growth patterns, and that proliferation of the cells is also affected by the concentration of matrix. In addition we found that 3D-cultures enhanced neuronal differentiation and the survival rate of the cells compared to 2D-cultures., Conclusions: Taken together, we have demonstrated a direct influence of the 3D-scaffold formation on the survival and neuronal differentiation of human neural progenitor cells. These findings emphasize the importance of optimizing 3D-scaffolds protocols prior to in vivo engraftment of stem and progenitor cells in the context of regenerative medicine.

Background & Aims: The family of serrated lesions includes hyperplastic polyps and sessile serrated adenomas without dysplasia, as well as traditional serrated adenoma with dysplasia. We investigated whether detection of proximal nondysplastic serrated polyps (ND-SP) at screening and surveillance colonoscopies is associated with advanced neoplasia., Methods: The study included 3121 asymptomatic patients (aged 50-75 years) who had screening colonoscopies; 1371 had subsequent surveillance. The proximal colon was defined as segments proximal to the descending colon. Large ND-SP were defined as ≥ 10 mm. We compared rates of detection of any neoplasia and advanced neoplasia at screening and surveillance colonoscopies (within 5.5 years) in patients with and without proximal or large ND-SP., Results: At baseline screening, 248 patients (7.9%) had at least 1 proximal ND-SP. They were more likely than patients with no proximal ND-SP to have advanced neoplasia (17.3% vs 10.0%; odds ratio [OR], 1.90; 95% confidence interval [CI], 1.33-2.70). Patients with large ND-SP (n = 44) were also more likely to have synchronous advanced neoplasia (OR, 3.37; 95% CI, 1.71-6.65). During surveillance, 39 patients with baseline proximal ND-SP and no neoplasia were more likely to have neoplasia compared with subjects who did not have polyps (OR, 3.14; 95% CI, 1.59-6.20). Among patients with advanced neoplasia at baseline, those with proximal ND-SP (n = 43) were more likely to have advanced neoplasia during surveillance (OR, 2.17; 95% CI, 1.03-4.59)., Conclusions: Detection of proximal and large ND-SP at a screening colonoscopy is associated with an increased risk for synchronous advanced neoplasia. Detection of proximal ND-SP in a baseline colonoscopy is associated with an increased risk for interval neoplasia during surveillance., (Copyright © 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: Nanomaterials are extensively used in industry and daily life, but little is known about possible health effects. An intensified research regarding toxicity of nanomaterials is urgently needed. Several studies have demonstrated that nanoparticles (NPs; diameter < 100 nm) can be transported to the central nervous system; however, interference of NPs with the electrical activity of neurons has not yet been shown., Objectives/methods: We investigated the acute electrophysiological effects of carbon black (CB), hematite (Fe2O3), and titanium dioxide (TiO2) NPs in primary murine cortical networks on microelectrode array (MEA) neurochips. Uptake of NPs was studied by transmission electron microscopy (TEM), and intracellular formation of reactive oxygen species (ROS) was studied by flow cytometry., Results: The multiparametric assessment of electrical activity changes caused by the NPs revealed an NP-specific and concentration-dependent inhibition of the firing patterns. The number of action potentials and the frequency of their patterns (spike and burst rates) showed a significant particle-dependent decrease and significant differences in potency. Further, we detected the uptake of CB, Fe2O3, and TiO2 into glial cells and neurons by TEM. Additionally, 24 hr exposure to TiO2 NPs caused intracellular formation of ROS in neuronal and glial cells, whereas exposure to CB and Fe2O3 NPs up to a concentration of 10 µg/cm2 did not induce significant changes in free radical levels., Conclusion: NPs at low particle concentrations are able to exhibit a neurotoxic effect by disturbing the electrical activity of neuronal networks, but the underlying mechanisms depend on the particle type.

The receptors engaged during recognition and phagocytic uptake of microorganisms and particles influence signaling events and diverse subcellular responses that occur during phagosome formation and maturation. However, pathogens generally have multiple ligands on their surface, making it difficult to dissect the roles of individual receptors during phagocytosis. Moreover, it remains elusive to which extent receptor-ligand interactions and early binding events define the subsequent intracellular fate of phagosomes. Here, we used latex beads coupled to single ligands, focusing on immunoglobulin G, mannan, bacterial lipopolysaccharides and avidin, and monitored: (1) phagocytic uptake rates, (2) fusion of phagosomes with lysosomal compartments, (3) the gene expression profile during phagocytosis, (4) the protein composition of mature phagosomes and (5) time-dependent dynamics of protein association with phagosomes in J774.A1 mouse macrophages. The differently coated latex beads were internalized at different rates and exhibited different kinetics of phagolysosomal fusion events dependent on their specific ligand. Furthermore, less than 60% of identified phagosomal proteins and only 10-15% of changes in gene expression were common to all investigated ligands. These findings demonstrate that each single ligand induced a distinct pattern of genes and a different protein composition of phagosomes. Taken together, our data argue that phagocytic receptor-specific programs of signaling events direct phagosomes to different physiological states and support the existence of a specific receptor-ligand 'signature' during the whole process of phagocytosis., (Copyright 2010 Elsevier GmbH. All rights reserved.)

Objectives: Withdrawal time (WT) has been proposed as a quality indicator for colonoscopy based on evidence that it is directly related to the rate of adenoma detection. Our objective was to test the hypothesis that baseline WT is inversely associated with the risk of finding neoplasia at interval colonoscopy., Methods: In all, 3,121 subjects, aged 50-75 years, had screening colonoscopy between 1994 and 1997 at 13 Veteran Affairs Medical Centers. In all, 1,193 subjects returned by protocol for surveillance within 5.5 years. In the 304 patients without polyps at baseline, we evaluated the contribution of baseline WT to their risk of interval neoplasia using bivariate and logistic regression analysis. We also examined the correlation between mean WT, baseline adenoma detection rate, and interval neoplasia rate at the medical-center level., Results: The average WT at the baseline exam in subjects with neoplasia on follow-up was 15.3 min as compared with 13.2 min in subjects without neoplasia (P=0.18). In a logistic regression model, WT was not associated with the risk of interval neoplasia (P=0.07). At the medical-center level, mean WT was not correlated with the probability of finding interval neoplasia (P=0.61) but was positively correlated with adenoma detection rate at baseline (P=0.03)., Conclusions: In this study with a mean baseline WT &12 min, there was no detectable association between WT and risk of future neoplasia. The medical center-level WT was positively correlated with adenoma detection. Therefore, above a certain threshold, WT may no longer be an adequate quality measure for screening colonoscopy.

Microelectrode arrays (MEAs) have been in use over the past decade and a half to study multiple aspects of electrically excitable cells. In particular, MEAs have been applied to explore the pharmacological and toxicological effects of numerous compounds on spontaneous activity of neuronal and cardiac cell networks. The MEA system enables simultaneous extracellular recordings from multiple sites in the network in real time, increasing spatial resolution and thereby providing a robust measure of network activity. The simultaneous gathering of action potential and field potential data over long periods of time allows the monitoring of network functions that arise from the interaction of all cellular mechanisms responsible for spatio-temporal pattern generation. In these functional, dynamic systems, physical, chemical, and pharmacological perturbations are holistically reflected by the tissue responses. Such features make MEA technology well suited for the screening of compounds of interest, and also allow scaling to high throughput systems that can record from multiple, separate cell networks simultaneously in multi-well chips or plates. This article is designed to be useful to newcomers to this technology as well as those who are currently using MEAs in their research. It explains how MEA systems operate, summarizes what systems are available, and provides a discussion of emerging mathematical schemes that can be used for a rapid classification of drug or chemical effects. Current efforts that will expand this technology to an influential, high throughput, electrophysiological approach for reliable determinations of compound toxicity are also described and a comprehensive review of toxicological publications using MEAs is provided as an appendix to this publication. Overall, this article highlights the benefits and promise of MEA technology as a high throughput, rapid screening method for toxicity testing., ((c) 2010 Elsevier Inc. All rights reserved.)

A series of novel 1,3,4-oxadiazole derivatives based on structural and electronic overlap with combretastatins have been designed and synthesized. Initially, we tested all new compounds in vivo using the phenotypic sea urchin embryo assay to yield a number of agents with anti-proliferative, anti-mitotic, and microtubule destabilizing activities. The experimental data led to identification of 1,3,4-oxadiazole derivatives with isothiazole (5-8) and phenyl (9-12) pharmacophores featuring activity profiles comparable to that of combretastatins, podophyllotoxin and nocodazole. Cytotoxic effects of the two lead molecules, namely 6 and 12, were further confirmed and evaluated by conventional assays with the A549 human cancer cell line including cell proliferation, cell cycle arrest at the G2/M phase, cellular microtubule distribution, and finally in vitro microtubule assembly with purified tubulin. The modeling results using 3D similarity (ROCS) and docking (FRED) correlated well with the observed activity of the molecules. Docking data suggested that the most potent molecules are likely to target the colchicine binding site., (Copyright (c) 2010 Elsevier Masson SAS. All rights reserved.)

Several adamantane-based taxol mimetics were synthesized and found to be cytotoxic at micromolar concentrations and to cause tubulin aggregation. The extent of the aggregation is maximal for N-benzoyl-(2R,3S)-phenylisoseryloxyadamantane (5) and is very sensitive to the structural modifications. A hybrid compound (15), combining adamantane-based taxol mimetic with colchicine was synthesized and found to possess both microtubule depolymerizing and microtubule bundling activities in A549 human lung carcinoma cells.

Background: Our aim was to identify markers predictive of fibrosis in alcoholic liver disease (ALD). Percutaneous liver biopsy is the recommended standard for histologic assessment of liver fibrosis. Seven serum markers (tissue inhibitor of matrix metalloproteinase 1 [TIMP1], tenascin, collagen VI, amino-terminal propeptide of type III collagen [PIIINP], matrix metalloproteinases [MMP2], laminin, and hyaluronic acid [HA]) representing various aspects of collagen and extracellular matrix deposition and degradation, have been proposed as noninvasive surrogates for liver biopsy. Moreover, a diagnostic algorithm including 3 serum markers (TIMP1, PIIINP, HA) and age has been proposed to accurately detect fibrosis with acceptable levels of sensitivity/specificity in a chronic hepatitis C subgroup., Methods: To determine variability of these markers in liver fibrosis with different etiologies, we conducted an evaluation of their correlative properties in a subgroup of patients (n = 247) with biopsy confirmed liver fibrosis resulting from long-term heavy alcohol consumption. Patients were participants in a recently completed VA multicenter clinical trial followed over 2 years with liver biopsy at baseline and 24 months, and with markers assessed every 3 months., Results: Among the markers measured in this alcoholic subgroup all except collagen VI displayed significant correlation with degrees of fibrosis. Three markers, TIMP1, PIIINP and HA adjusted for age, emerged as the most promising predictors of the degree of fibrosis in a population of alcoholics. However, there was little change over time as related to change in fibrosis. The lower than expected accuracy of these markers based on receiver operating curves (ROC) also showed their limited use in this etiologic subgroup., Conclusion: In alcoholic patients, various markers have limited value in predicting and diagnosing the stages of fibrosis compared to liver biopsy. Thus, further prospective studies are required to better define the usefulness of each marker or their combination which are possibly affected by alcohol metabolism.

Interphase microtubules are organized into a radial array with centrosome in the center. This organization is a subject of cellular regulation that can be driven by protein phosphorylation. Only few protein kinases that regulate microtubule array in interphase cells have been described. Ste20-like protein kinase LOSK (SLK) was identified as a microtubule and centrosome-associated protein. In this study we have shown that the inhibition of LOSK activity by dominant-negative mutant K63R-DeltaT or by LOSK depletion with RNAi leads to unfocused microtubule arrangement. Microtubule disorganization is prominent in Vero, CV-1, and CHO-K1 cells but less distinct in HeLa cells. The effect is a result neither of microtubule stabilization nor of centrosome disruption. In cells with suppressed LOSK activity centrosomes are unable to anchor or to cap microtubules, though they keep nucleating microtubules. These centrosomes are depleted of dynactin. Vero cells overexpressing K63R-DeltaT have normal dynactin "comets" at microtubule ends and unaltered morphology of Golgi complex but are unable to polarize it at the wound edge. We conclude that protein kinase LOSK is required for radial microtubule organization and for the proper localization of Golgi complex in various cell types.

Traditional Parkinson's disease models in rats have several disadvantages. A promising alternative in terms of a more physiological model was proposed by McNaught et al. [McNaught, K.S., Perl, D.P., Brownell, A.L., Olanow, C.W., 2004. Systemic exposure to proteasome inhibitors causes a progressive model of Parkinson's disease. Ann. Neurol. 56, 149-162.] inhibiting the proteasomal protein degradation in vivo where they observed in Sprague-Dawley rats distinct symptoms of Parkinson's disease, a typical slow progredient loss of dopaminergic neurons in the substantia nigra and a lack of dopaminergic afferences in the striatum. We administered to Wistar rats a synthetic proteasome inhibitor (PSI) analogous to the published method. Locomotor changes were analysed by a footprint test. Brain slices containing the substantia nigra and the striatum were stained immunohistochemically against tyrosine hydroxylase, neuronal nuclei antigen, glial fibrillary acidic protein, alpha-synuclein and microglia. Standard histological stainings (haematoxylin eosin or Nissl) were also performed. The proteasome inhibitor effect on the glomerular layer of the olfactory bulb, the adrenal medulla and the carotid body was examined. We observed no PSI-induced motor deficits and loss of tyrosine hydroxylase immunoreactivity in the substantia nigra or the striatum. However, we detected a distinct increase of tyrosine hydroxylase immunoreactivity in the glomerular layer of the olfactory bulb and in the adrenal medulla. Our results fall in line with reports of other research groups which failed to reproduce the original report, but here for the first time McNaughts model could not be reproduced in Wistar rats. The observed effects on the olfactory bulb and peripheral catecholaminergic organs speak for an impermeability of the blood brain barrier for PSI.

Background & Aims: Outcomes of colon surveillance after colorectal cancer screening with colonoscopy are uncertain. We conducted a prospective study to measure incidence of advanced neoplasia in patients within 5.5 years of screening colonoscopy., Methods: Three thousand one hundred twenty-one asymptomatic subjects, age 50 to 75 years, had screening colonoscopy between 1994 and 1997 in the Department of Veterans Affairs. One thousand one hundred seventy-one subjects with neoplasia and 501 neoplasia-free controls were assigned to colonoscopic surveillance over 5 years. Cohorts were defined by baseline findings. Relative risks for advanced neoplasia within 5.5 years were calculated. Advanced neoplasia was defined as tubular adenoma greater than > or =10 mm, adenoma with villous histology, adenoma with high-grade dysplasia, or invasive cancer., Results: Eight hundred ninety-five (76.4%) patients with neoplasia and 298 subjects (59.5%) without neoplasia at baseline had colonoscopy within 5.5 years; 2.4% of patients with no neoplasia had interval advanced neoplasia. The relative risk in patients with baseline neoplasia was 1.92 (95% CI: 0.83-4.42) with 1 or 2 tubular adenomas <10 mm, 5.01 (95% CI: 2.10-11.96) with 3 or more tubular adenomas <10 mm, 6.40 (95% CI: 2.74-14.94) with tubular adenoma > or =10 mm, 6.05 (95% CI: 2.48-14.71) for villous adenoma, and 6.87 (95% CI: 2.61-18.07) for adenoma with high-grade dysplasia., Conclusions: There is a strong association between results of baseline screening colonoscopy and rate of serious incident lesions during 5.5 years of surveillance. Patients with 1 or 2 tubular adenomas less than 10 mm represent a low-risk group compared with other patients with colon neoplasia.

The possible harmful effects of radiofrequency electromagnetic fields (RF EMFs) are controversial. We have used human Mono Mac 6 cells to investigate the influence of RF EMFs in vitro on cell cycle alterations and BrdU uptake, as well as the induction of apoptosis and necrosis in human Mono Mac 6 cells, using flow cytometry after exposure to a 1,800 MHz, 2 W/kg specific absorption rate (SAR), GSM-DTX signal for 12 h. No statistically significant differences in the induction of apoptosis or necrosis, cell cycle kinetics, or BrdU uptake were detected after RF EMF exposure compared to sham or incubator controls. However, in the positive control cells treated with gliotoxin and PMA (phorbol 12 myristate-13 acetate), a significant increase in apoptotic and necrotic cells was seen. Cell cycle analysis or BrdU incorporation for 72 h showed no differences between RF EMF- or sham-exposed cells, whereas PMA treatment induced a significant accumulation of cells in G(0)/G(1)-phase and a reduction in S-phase cells. RF EMF radiation did not induce cell cycle alterations or changes in BrdU incorporation or induce apoptosis and necrosis in Mono Mac 6 cells under the exposure conditions used.

The aim of the present study is to investigate whether extremely low frequency electromagnetic fields (ELF-EMF) affect certain cellular functions and immunologic parameters of mouse macrophages. In this study, the influence of 50 Hz magnetic fields (MF) at 1.0 mT was investigated on the phagocytic activity and on the interleukin-1beta (IL-1beta) production in differentiated macrophages. MF-exposure led to an increased phagocytic activity after 45 min, shown as a 1.6-fold increased uptake of latex beads in MF-exposed cells compared to controls. We also demonstrate an increased IL-1beta release in macrophages after 24 h exposure (1.0 mT MF). Time-dependent IL-1beta formation was significantly increased already after 4 h and reached a maximum of 12.3-fold increase after 24 h compared to controls. Another aspect of this study was to examine the genotoxic capacity of 1.0 mT MF by analyzing the micronucleus (MN) formation in long-term (12, 24, and 48 h) exposed macrophages. Our data show no significant differences in MN formation or irregular mitotic activities in exposed cells. Furthermore, the effects of different flux densities (ranging from 0.05 up to 1.0 mT for 45 min) of 50 Hz MF was tested on free radical formation as an endpoint of cell activation in mouse macrophage precursor cells. All tested flux densities significantly stimulated the formation of free radicals. Here, we demonstrate the capacity of ELF-EMF to stimulate physiological cell functions in mouse macrophages shown by the significantly elevated phagocytic activity, free radical release, and IL-1beta production suggesting the cell activation capacity of ELF-EMF in the absence of any genotoxic effects.

Objective: Aspartate aminotransferase (AST) to platelet ratio index (APRI) has been proposed as an easily determined and accurate noninvasive marker of liver fibrosis in chronic hepatitis C. To validate APRI in hepatitis C and to determine its usefulness in other liver diseases, we evaluated APRI in patients with liver fibrosis due to excessive alcohol consumption with or without viral hepatitis C., Methods: A total of 1,308 subjects from two VA cooperative studies of alcoholic liver disease were evaluated. Liver biopsy was available from 781 noncirrhotic patients while a history of decompensation was present in 527. Alcohol intake was determined by self-report. Hepatitis C was confirmed by PCR., Results: Ninety-eight percent were men with a mean age of 51.5 yr. Alcohol intake averaged 19 drinks/day for 20.6 yr. One hundred thirty-three (10.2%) were hepatitis C positive. In the HCV-positive subgroup, APRI had a sensitivity of 35.6% and a specificity of 29.7% for significant fibrosis. Of 64 patients classified as significant fibrosis, 21 (32.8%) were incorrectly classified. In the 507 HCV negative patients with biopsy confirmed fibrosis, the sensitivity of APRI for significant fibrosis was 13.2% and the specificity was 77.6%. Twenty percent were classified incorrectly., Conclusion: APRI has low sensitivity and specificity for the diagnosis of significant fibrosis in patients with alcoholic liver disease, including patients who have hepatitis C. Given the frequent history of alcohol use in patients with hepatitis C, APRI may be of limited usefulness in the diagnosis of fibrosis in many patients.

We optimized the novel technique of multielectrode neurochip recordings for the rapid and efficient screening of neuroactivity. Changes in the spontaneous activity of cultured networks of primary cortical neurons were quantified to evaluate the action of drugs on the firing dynamics of complex network activity. The multiparametric assessment of electrical activity changes caused by psychoactive herbal extracts from Hypericum, Passiflora and Valeriana, and various combinations thereof revealed a receptor-specific and concentration-dependent inhibition of the firing patterns. The spike and burst rates showed significant substance-dependent effects and significant differences in potency. The effects of specific receptor blockades on the inhibitory responses provided evidence that the herbal extracts act on gamma-amino butyric acid (GABA) and serotonin (5-HT) receptors, which are recognized targets of pharmacological antidepressant treatment. A biphasic effect, serotonergic stimulation of activity at low concentrations that is overridden by GABAergic inhibition at higher concentrations, is apparent with Hypericum alone and the triple combination of the extracts. The more potent neuroactivity of the triple combination compared to Hypericum alone and the additive effect of Passiflora and Valeriana suggest a synergy between constituent herbal extracts. The extracts and their combinations affected the set of derived activity parameters in a concomitant manner suggesting that all three constituent extracts and their combinations have largely similar modes of action. This study also demonstrates the sensitivity, selectivity and robustness of neurochip recordings for high content screening of complex mixtures of neuroactive substances and for providing multiparametric information on neuronal activity changes to assess the therapeutic potential of psychoactive substances.

This study was conducted to investigate the potential for increased oxidative stress of high- vs. average-producing dairy cows. Two experiments were performed using 11 and 13 Holstein cows (53 +/- 2 d postpartum). Lipohydroperoxides (LHP) were determined in serum lipids (experiment 1) and low-density lipoprotein (experiment 2) via oxidation of ferrous to ferric ions through LHP using thiocyanate as chromogen. In experiment 1, differing milk yield and milk energy output corresponded to different concentrations of LHP. In experiment 2, analysis of regression resulted in a significant relationship between milk yield and LHP. Phospholipids isolated from lipids with 6.5 microM of LHP evoked in monocytic cells a transient increase in superoxide formation, indicating inflammatory potential. The results show that high milk productivity can associate with oxidative stress indicated by oxidative modifications of circulating lipids and their changed bioactivity.

Background & Aims: Colchicine improved survival and reversed cirrhosis in several small clinical trials. We compared the efficacy and safety of long-term colchicine, as compared with placebo, in patients with advanced alcoholic cirrhosis., Methods: Five hundred forty-nine patients with advanced (Pugh B or C) alcoholic cirrhosis were randomized to receive either colchicine 0.6 mg twice per day (n = 274) or placebo (n = 275). Treatment lasted from 2 to 6 years. The primary outcome was all-cause mortality. Secondary outcomes were liver-related morbidity and mortality. Liver biopsy was requested prior to entry and after 24 months of treatment., Results: Attendance at scheduled clinic visits and adherence with study medication were similar in colchicine and placebo groups. Alcohol intake was less than 1 drink per day in 69% of patients. In an intention-to-treat analysis, all-cause mortality was similar in colchicine (49%) and placebo (45%) patients (P = .371). Mortality attributed to liver disease was 32% in colchicine and 28% in placebo patients (P = .337). Fewer patients receiving colchicine developed hepatorenal syndrome. In 54 patients with repeat liver biopsies after 24 or more months of treatment, cirrhosis improved to septal fibrosis in 7 patients (3 colchicine, 4 placebo) and to portal fibrosis in 1 patient (colchicine)., Conclusions: In patients with advanced alcoholic cirrhosis, colchicine does not reduce overall or liver-specific mortality. Liver histology improves to septal fibrosis in a minority of patients after 24 months of treatment, with similar rates of improvement in patients receiving placebo and colchicine. Colchicine is not recommended for patients with advanced alcoholic cirrhosis.

Objective: To study the biological effectiveness of Auger electrons emitted by (99m)Tc on cell survival, induction of apoptosis and micronucleus (MN) formation in the human squamous cell carcinoma cell line SCL-II and compare the effects observed to those observed after exposure to external 60Co gamma radiation., Material and Methods: Cells were either gamma(60Co)-irradiated (0.67 Gy/min) or exposed to (99m)Tc-pertechnetate (0.95-14.3 MBq/ml) for 24 h under cell culture conditions and assayed for cell survival (colony-forming assay), micronucleus formation (cytochalasin B assay) and the frequency of apoptotic cells (fluorescence microscopy). Monte Carlo based dosimetry has been applied to derive the absorbed dose corresponding to the accumulated decays of (99m)Tc under the given geometry., Results: Absorbed doses up to 0.5 Gy could be achieved after 99mTc-exposure leading to no substantial cell killing in this dose range except at one dose point (0.1 Gy) resulting in an relative biological effectiveness (RBE)SF 0.9 of 0.64 when compared to the 60Co reference radiation. MN formation was described best by a linear dose response and was consistently lower after 99mTc exposure when compared to 60Co irradiated cells resulting in an RBE of 0.37. Apoptosis induction was significantly increased after 99mTc exposure at much lower doses (0.1 Gy) when compared to the reference radiation. The (99m)Tc uptake experiments revealed an activity concentration ratio cells vs. medium of 0.07 after 24 h of exposure., Conclusion: No overall increased biological effectiveness due to the emitted Auger electrons of (99m)Tc, applied as sodium-pertechnetate, could be observed in the investigated cell line when compared to acute external gamma radiation. The RBEs in the range of 0.37-0.64 might be well explained by dose rate effects. The significantly increased apoptotic response after (99m)Tc-exposure at very low doses has to be further investigated.

Cell survival, induction of apoptosis, and micronucleus formation have been examined in non-transformed human amnion fluid fibroblast-like (AFFL) cells and in a human squameous cell carcinoma (SCL-II) cell line after exposure to the Auger electron emitter (65)Zn and after external low-LET radiation. Cellular uptake and subcellular distribution of (65)Zn(2+) were studied in vitro and the absorbed radiation dose was calculated applying analytical dosimetry models. Auger electrons generated during decay of (65)Zn induced a prominent decrease in cell survival and increased the levels of apoptotic as well as micronucleated cells when compared to external low-LET irradiation. Relative biological effectiveness has been determined for cell survival (RBE approximately 4), micronucleus formation (RBE approximately 2) and apoptosis induction (RBE approximately 5-8) in SCL-II cells and for micronucleus formation (RBE approximately 4-5) and apoptosis induction (RBE approximately 6-10) in AFFL cells, respectively. This demonstrates a general enhanced biological effectiveness of (65)Zn in both investigated cell lines when compared to external low-LET radiation. The distribution pattern of intracellular Zn(2+) was found to be non-uniform, showing enhanced amounts of Zn(2+) in the perinuclear region and low amounts inside the cell nucleus, suggesting a major energy deposition close to the nuclear envelope.

This paper presents a novel and comprehensive method to identify substances on the basis of electrical activity and is a substantial improvement for drug screening. The spontaneous activity of primary neuronal networks is influenced by neurotransmitters, ligands, and other substances in a similar fashion as known from in vivo pharmacology. However, quantitative methods for the identification of substances through their characteristic effects on network activity states have not yet been reported. We approached this problem by creating a database including native activity and five drug-induced oscillatory activity states from extracellular multisite recordings from microelectrode arrays. The response profiles consisted of 30 activity features derived from the temporal distribution of action potentials, integrated burst properties, calculated coefficients of variation, and features of Gabor fits to autocorrelograms. The different oscillatory states were induced by blocking neurotransmitter receptors for: (i) GABA(A); (ii) glycine; (iii) GABA(A) and glycine; (iv) all major synaptic types except AMPA, and (v) all major synapses except NMDA. To test the identification capability of the six substance-specific response profiles, five blind experiments were performed. The response features from the unknown substances were compared to the database using proximity measures using the normalized Euclidian distance to each activity state. This process created six identification coefficients where the smallest correctly identified the unknown substances. Such activity profiles are expected to become substance-specific 'finger prints' that classify unique responses to known and unknown substances. It is anticipated that this kind of approach will help to quantify pharmacological responses of networks used as biosensors.

The genotoxicity of asbestos fibers is generally mediated by reactive oxygen species (ROS) and by insufficient antioxidant protection. To further elucidate which radicals are involved in asbestos-mediated genotoxicity and to which extent, we have carried out experiments with the metal chelators deferoxamine (DEF) and phytic acid (PA), and with the radical scavengers superoxide dismutase (SOD), dimethylthiourea (DMTU) and the glutathione precursor Nacystelyn trade mark (NAL). We investigated the influence of these compounds on the potency of crocidolite, an amphibole asbestos fiber with a high iron content (27%), and chrysotile, a serpentine asbestos fiber with a low iron content (2%), to induce micronuclei (MN) in human mesothelial cells (HMC) after an exposure time of 24-72 h. Our results show that the number of crocidolite-induced MN is significantly reduced after pretreatment of fibers with PA and DEF. This effect was not observed with chrysotile. In contrast, simultaneous treatment of cells with asbestos and the OH*scavenging DMTU or the O2- -scavenging SOD significantly decreased the number of MN induced by chrysotile and crocidolite. In particular, DMTU almost completely suppressed micronucleus induction by both fiber types. A similar effect was observed in the presence of the H(2)O(2)-scavenging NAL after chrysotile treatment of HMC. By means of kinetochore analysis, it could be shown that the number of clastogenic events is decreased after PA and DEF pretreatment of fibers as well as after application of the above-mentioned scavengers. Our results show that chrysotile asbestos induces an increased release of H(2)O(2) in contrast to crocidolite. Also, the iron content of the fiber plays an important role in radical formation, but nevertheless, chrysotile produces oxy radicals to a similar extent as crocidolite, probably by phagocytosis-mediated oxidative bursting.

Context: Knowledge of risk factors for colorectal neoplasia could inform risk reduction strategies for asymptomatic individuals. Few studies have evaluated risk factors for advanced colorectal neoplasia in asymptomatic individuals, compared risk factors between persons with and without polyps, or included most purported risk factors in a multivariate analysis., Objective: To determine risk factors associated with advanced colorectal neoplasia in a cohort of asymptomatic persons with complete colonoscopy., Design, Setting, and Participants: Prospective, cross-sectional study of 3121 asymptomatic patients aged 50 to 75 years from 13 Veterans Affairs medical centers conducted between February 1994 and January 1997. All participants had complete colonoscopy to determine the prevalence of advanced neoplasia, defined as an adenoma that was 10 mm or more in diameter, a villous adenoma, an adenoma with high-grade dysplasia, or invasive cancer. Variables examined included history of first-degree relative with colorectal cancer, prior cholecystectomy, serum cholesterol level, physical activity, smoking, alcohol use, and dietary factors., Main Outcome Measures: An age-adjusted analysis was performed for each variable to calculate the odds ratios (ORs) and 95% confidence intervals (CIs) associated with having advanced neoplasia compared with having no polyps. We developed a multivariate logistic regression model to identify the most informative risk factors. A secondary analysis examined risk factors for having hyperplastic polyps compared with having no polyps and compared with having advanced neoplasia., Results: Three hundred twenty-nine participants had advanced neoplasia and 1441 had no polyps. In multivariate analyses, we found positive associations for history of a first-degree relative with colorectal cancer (OR, 1.66; 95% CI, 1.16-2.35), current smoking (OR, 1.85; 95% CI, 1.33-2.58), and current moderate to heavy alcohol use (OR, 1.02; 95% CI, 1.01-1.03). Inverse associations were found for cereal fiber intake (OR, 0.95; 95% CI, 0.91-0.99), vitamin D intake (OR, 0.94; 95% CI, 0.90-0.99), and use of nonsteroidal anti-inflammatory drugs (NSAIDs) (OR, 0.66; 95% CI, 0.48-0.91). In the univariate analysis, the inverse association was found with cereal fiber intake greater than 4.2 g/d, vitamin D intake greater than 645 IU/d, and daily use of NSAIDs. Marginal factors included physical activity, daily multivitamin use, and intake of calcium and fat derived from red meat. No association was found for body mass index, prior cholecystectomy, or serum cholesterol level. Three hundred ninety-one patients had hyperplastic polyps as the worst lesion found at colonoscopy. Risk variables were similar to those for patients with no polyps, except that past and current smoking were associated with an increased risk of hyperplastic polyps., Conclusions: Our data endorse several important risk factors for advanced colonic neoplasia and provide a rationale for prudent risk reduction strategies. Further study is needed to determine if lifestyle changes can moderate the risk of colorectal cancer.