Vitamin E Protects DNA from Oxidative Damage in Human Hepatocellular Carcinoma Cell Lines

Expression of multiple drug resistant (MDR) phenotype and over-expression of P-glycoprotein (P-gp) in the human hepatocellular carcinoma (HCC) cell clone P1(0.5), derived from the PLC/PRF/5 cell line (P5), are associated with strong resistance to oxidative stress and a significant (?p<0.01) increase in intracellular vitamin E content as compared with the parental cell line. This study evaluates the role of vitamin E in conferring resistance to drugs and oxidative stress in P1(0.5) cells. Parental drug-sensitive cells, P5, were incubated in α-tocopherol succinate (α-TS, 5?μM for 24?h) enriched medium to increase intracellular vitamin E content to levels comparable to those observed in P1(0.5) cells at basal conditions. Susceptibility to lipid peroxidation and oxidative DNA damage were assessed by measuring the concentration of thiobarbituric-reactive substances (TBARS) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) at basal and after experimental conditions. Cell capacity to form colonies and resistance to doxorubicin were also studied. P5 cells, treated with α-TS, became resistant to ADP-Fe³⁺ and to ionizing radiation-induced lipid peroxidation as P1(0.5) cells. Exposure to ADP-Fe³⁺ or ionizing radiation increased TBARS and the 8-OHdG content in the P5 cells, while vitamin E enrichment abolished these effects. Irradiation doses at 5?cGy increased TBARS and 8-OHdG. They also inhibited cell capacity to form colonies in the untreated P5 cells. Incubation with α-TS fully reverted this effect and significantly (p<0.01) reduced the inhibitory effect of cell proliferation induced by irradiation doses at >500?cGy. Resistance to doxorubicin was not affected by α-TS. These observations demonstrate the role of vitamin E in conferring protection from lipid peroxidation, ionizing radiation and oxidative DNA damage on the human HCC cell line. They also rule out any role of P-gp over-expression as being responsible for these observations in cells with MDR phenotype expression.     

Neuropsychopathological comorbidities in learning disorders

Background

Learning Disorders (LD) are complex diseases that affect about 2-10% of the school-age population. We performed neuropsychological and psychopathological evaluation, in order to investigate comorbidity in children with LD.

Methods

Our sample consisted of 448 patients from 7 to 16 years of age with a diagnosis of LD, divided in two subgroups: Specific Learning Disorders (SLD), including reading, writing, mathematics disorders, and Learning Disorders Not Otherwise Specified (LD NOS).

Results

Comorbidity with neuropsychopathologies was found in 62.2% of the total sample. In the LSD subgroup, ADHD was present in 33%, Anxiety Disorder in 28.8%, Developmental Coordination Disorder in 17.8%, Language Disorder in 11% and Mood Disorder in 9.4% of patients. In LD NOS subgroup, Language Disorder was present in 28.6%, Developmental Coordination Disorder in 27.5%, ADHD in 25.4%, Anxiety Disorder in 16.4%, Mood Disorder in 2.1% of patients. A statistically significant presence was respectively found for Language and Developmental Coordination Disorder comorbidity in LD NOS and for ADHD, mood and anxiety disorder comorbidity in SLD subgroup.

Conclusions

The different findings emerging in this study suggested to promote further investigations to better define the difference between SLD and LD NOS, in order to improve specific interventions to reduce the long range consequences.

     

Role Preferences of People with Multiple Sclerosis: Image-Revised,Computerized Self-Administered Version of the Control Preference Scale

Background

The Control Preference Scale (CPS) is the most frequently used measure of patients’ preferred roles in treatment decisions. We revised the original CPS and developed a new computerized patient self-administered version (eCPS). We used the eCPS to assess role preferences, and their determinants, in Italian and German people with multiple sclerosis (MS).

Methods

New cartoons were produced, based on MS health professional and patient input/feedback and previous findings, and pilot tested on 26 Italian and German MS patients. eCPS acceptability and reliability (weighted kappa statistic, wK) in comparison to the original tool, was determined in 92 MS patients who received both CPS versions in random order.

Results

The new cartoons were well accepted and easily interpreted by patients, who reported they based their choices mainly on the text and considered the images of secondary importance. eCPS reliability was moderate (wK 0.53, 95% confidence interval [CI] 0.40–0.65) and similar to the test-retest reliability of face-to-face administration assessed in a previous publication (wK 0.65, 95% CI 0.45–0.81). Higher education (odds ratio [OR] 3.74, 95% CI 1.00–14.05) and German nationality (OR 10.30, 95% CI 3.10–34.15) were associated with preference for an active role in the logistic model.

Conclusions

The newly devised eCPS was well received and considered easy to use by MS patients. Reliability was in line with that of the original version. Role preference appears affected by cultural characteristics and (borderline statistical significance) education.     

TNFα Signals via p66Shc to Induce E-Selectin,Promote Leukocyte Transmigration and Enhance Permeability in Human Endothelial Cells

Endothelial cells participate in inflammatory events leading to atherogenesis by regulating endothelial cell permeability via the expression of VE-Cadherin and β-catenin and leukocyte recruitment via the expression of E-Selectins and other adhesion molecules. The protein p66^Shc acts as a sensor/inducer of oxidative stress and may promote vascular dysfunction. The objective of this study was to investigate the role of p66^Shc in tumor necrosis factor TNFα-induced E-Selectin expression and function in human umbilical vein endothelial cells (HUVEC). Exposure of HUVEC to 50 ng/ml TNFα resulted in increased leukocyte transmigration through the endothelial monolayer and E-Selectin expression, in association with augmented phosphorylation of both p66^Shc on Ser³⁶ and the stress kinase c-Jun NH₂-terminal protein kinase (JNK)-1/2, and higher intracellular reactive oxygen species (ROS) levels. Overexpression of p66^Shc in HUVEC resulted in enhanced p66^Shc phosphorylation on Ser³⁶, increased ROS and E-Selectin levels, and amplified endothelial cell permeability and leukocyte transmigration through the HUVEC monolayer. Conversely, overexpression of a phosphorylation-defective p66^Shc protein, in which Ser³⁶ was replaced by Ala, did not augment ROS and E-Selectin levels, nor modify cell permeability or leukocyte transmigration beyond those found in wild-type cells. Moreover, siRNA-mediated silencing of p66^Shc resulted in marked reduction of E-Selectin expression and leukocyte transmigration. In conclusion, p66^Shc acts as a novel intermediate in the TNFα pathway mediating endothelial dysfunction, and its action requires JNK-dependent phosphorylation of p66^Shc on Ser³⁶.     

Changes in paracrine interleukin-2 requirement, CCR7 expression, frequency, and cytokine secretion of human immunodeficiency virus-specific CD4+ T cells are a consequence of antigen load

Virus-specific CD4+ T-cell responses are thought to be required for the induction and maintenance of many effective CD8+ T-cell and B-cell immune responses in experimental animals and humans. Although the presence of human immunodeficiency virus (HIV)-specific CD4+ T cells has been documented in patients at all stages of HIV infection, many fundamental questions regarding their frequency and function remain. A 10-color, 12-parameter flow cytometric panel was utilized to examine the frequency, memory phenotype (CD27, CCR7, and CD45RA), and cytokine production (interleukin-2 [IL-2], gamma interferon, and tumor necrosis factor alpha) of CD4+ T cells specific for HIV antigens as well as for adenovirus, Epstein-Barr virus (EBV), influenza H1N1 virus, influenza H3N2 virus, cytomegalovirus, varicella-zoster virus (VZV), and tetanus toxoid in normal controls, long-term nonprogressors (LTNP), and HIV-infected patients with progressive disease on or off therapy. The HIV-specific CD4+ T-cell responses in LTNP and patients on therapy were similar in frequency, phenotype, and cytokine production to responses directed against adenovirus, EBV, influenza virus, and VZV. HIV-specific CD4+ T cells from patients off antiretroviral therapy demonstrated a shift towards a CCR7(-) CD45RA(-) phenotype and a reduced percentage of IL-2-producing cells. The alterations in cytokine production during HIV viremia were found to be intrinsic to the HIV-specific CD4+ T cells and caused a requirement for IL-2 supplied exogenously for proliferation to occur. These observations suggest that many previously described changes in HIV-specific CD4+ T-cell function and phenotype are a consequence of high levels of antigen in viremic patients. In addition, defects in function and phenotype of HIV-specific CD4+ T cells are not readily discernible in the context of antiretroviral therapy but rather are similar to responses to other viruses.     

Novel route for elimination of brain oxysterols across the blood-brain barrier: conversion into 7alpha-hydroxy-3-oxo-4-cholestenoic acid

Recently, we demonstrated a net blood-to-brain passage of the oxysterol 27-hydroxycholesterol corresponding to 4-5 mg/day. As the steady-state levels of this sterol are only 1-2 mug/g brain tissue, we hypothesized that it is metabolized and subsequently eliminated from the brain. To explore this concept, we first measured the capacity of in vitro systems representing the major cell populations found in the brain to metabolize 27-hydroxycholesterol. We show here that 27-hydroxycholesterol is metabolized into the known C(27) steroidal acid 7alpha-hydroxy-3-oxo-4-cholestenoic acid by neuronal cell models only. Using an in vitro model of the blood-brain barrier, we demonstrate that 7alpha-hydroxy-3-oxo-4-cholestenoic acid is efficiently transferred across monolayers of primary brain microvascular endothelial cells. Finally, we measured the concentration of 7alpha-hydroxy-3-oxo-4-cholestenoic acid in plasma from the internal jugular vein and brachial artery of healthy volunteers. Calculation of the arteriovenous concentration difference revealed a significant in vivo flux of this steroid from the brain into the circulation in human. Together, these studies identify a novel metabolic route for the elimination of 27-hydroxylated sterols from the brain. Given the emerging connections between cholesterol and neurodegeneration, this pathway may be of importance for the development of these conditions.     

Short-term effects of 3,4-methylen-dioxy-metamphetamine (MDMA) on 5-HT(1A) receptors in the rat hippocampus

The first effects of 3,4-methylen-dioxy-metamphetamine (MDMA, “ecstasy”), on serotonin 1A (5-HT_1A) receptors in rat hippocampus were determined by means of [³H]-8-hydroxy-dipropylamino-tetralin ([³H]-8-OH-DPAT) and 5′guanosine-(γ-[³⁵S]-thio)triphosphate ([³⁵S]-GTPγS) binding as well as inhibition of forskolin (FK)-stimulated adenylyl cyclase (AC) activity. The study was completed by [³⁵S]-GTPγS functional autoradiography experiments carried out in frontal sections of rat brain, including the hippocampal region. Results showed that MDMA was either able to displace [³H]-8-OH-DPAT binding (K_i  500 nM) or to reduce the number of specific sites (B_max) without affecting K_d. The drug also failed to change the [³⁵S]-GTPγS binding or to inhibit AC velocity, underlying its behavior as a non-competitive 5-HT_1A receptor antagonist. Further, MDMA (1 or 100 μM), partially antagonized either [³⁵S]-GTPγS binding stimulation of the agonists 5CT and 8-OH-DPAT or the AC inhibition induced by 5CT and DP-5CT. However, in contrast to binding studies, in AC assays the amphetamine displayed an effect also on EC₅₀, always being less potent than the reference antagonist WAY100,635. In functional autoradiography, MDMA behaved either as a partial 5-HT_1A antagonist in limbic areas or, added alone, as an agonist, increasing the coupling signal presumably through 5-HT release from synapses. Interestingly, the selective 5-HT re-uptake inhibitor (SSRI) fluoxetine had no effect on MDMA [³⁵S]-GTPγS binding activation. This latter finding indicates that the amphetamine can release 5-HT via alternative mechanisms to 5-HT transporter binding, probably via membrane synaptic receptors or vesicular transporters. The release of other transmitters is not excluded. Therefore, our results encourage at extending the study of MDMA biochemical profiles, in the attempt to elucidate those amphetamine-induced pathways with a potential for neurotoxicity or psycho-stimulant activity.     

Smokers and passive smokers gene expression profiles: correlation with the DNA oxidation damage

Healthy volunteers (n=50) were enrolled for studying the variation of gene expression induced by smoking in peripheral lymphocytes. RNAs from smokers (>3 cigarettes/day, n=20) and passive smokers (exposed to tobacco smoke >3 h/day, n=10) were hybridized versus a reference pool obtained by mixing equal amounts of RNA from 20 nonsmokers, and gene expression was analyzed using DNA microarrays containing 13,971 oligos. Principal component analysis showed that 99.7% of gene expression variability was related to plasma cotinine, age, and DNA oxidation damage. SAM and GenMAPP/MAPPFinder analyses showed that smokers, compared to nonsmokers, had 129 down-regulated and 87 up-regulated genes, whereas passive smokers, compared to nonsmokers, had 44 down-regulated and 159 up-regulated genes, mainly involved in pathways associated with the activation of defensive responses. Hierarchical cluster analysis identified two distinct clusters of smokers, characterized by different oxidative DNA damage: smokers with high DNA oxidation damage, compared to smokers with low DNA oxidation damage, had a large number (150) of down-regulated genes, mainly associated with xenobiotic metabolism, DNA damage and repair, inflammatory responses, lymphocyte activation, and cytokine activity, suggesting a reduced cellular response to toxic agents in this subset of smokers that could lead to an increased DNA oxidation damage.