The synthetic liver X receptor agonist GW3965 reduces tissue factor production and inflammatory responses in human islets in vitro.

AIMS/HYPOTHESIS: Optimising islet culture conditions may be one strategy for reducing islet loss prior to, and immediately after, islet transplantation. Liver X receptor (LXR) agonism has previously been shown to increase insulin release from pancreatic islets and reduce inflammation in leucocytes. Our aim was to investigate whether the synthetic LXR agonist GW3965 could modulate the inflammatory status of human pancreatic islets. METHODS: Levels of pro-inflammatory cytokines and tissue factor in isolated human islets were determined by TaqMan low density array and/or real-time quantitative RT-PCR (mRNA levels) and enzyme immunoassay (EIA) (protein levels). Islet viability was measured using intracellular ATP content, ADP/ATP ratio, mitochondrial dehydrogenase activity (XTT assay) and insulin secretion in a dynamic glucose-challenge model. Apoptosis was determined by EIA measurement of histone-DNA complexes present in cytoplasm and by assaying caspase-3/-7 activity. RESULTS: Treatment of LPS-stimulated human islets with the synthetic LXR agonist GW3965 (1 micromol/l) for 24 h reduced mRNA and protein levels of selected pro-inflammatory cytokines (IL-8, monocyte chemotactic protein-1 and tissue factor). Moreover, GW3965 had no adverse effect on insulin secretion, islet viability or apoptosis. No excess of lipid accumulation could be detected with the dosage and exposure time used. CONCLUSIONS/INTERPRETATION: LXR activation suppresses inflammation in human islets in vitro without adverse effects on islet viability. Short-term moderate activation of LXR prior to islet transplantation may represent a possible strategy for improving post-transplant islet survival.

A novel technique for fascial fixation of laparoscopic adjustable gastric band ports.

Access port dislodgement after laparoscopic adjustable gastric banding is a recurring problem that often requires operative revision. Securing the port to the abdominal wall fascia in the traditional way with standard instruments is challenging in obese patients due to a thick abdominal wall. Therefore, we have devised a novel and simple technique for access port fixation using the EndoStitch device.

Structural determinants of ligand binding selectivity between the peroxisome proliferator-activated receptors.

The peroxisome proliferator-activated receptors (PPARs) are transcriptional regulators of glucose, lipid, and cholesterol metabolism. We report the x-ray crystal structure of the ligand binding domain of PPAR alpha (NR1C1) as a complex with the agonist ligand GW409544 and a coactivator motif from the steroid receptor coactivator 1. Through comparison of the crystal structures of the ligand binding domains of the three human PPARs, we have identified molecular determinants of subtype selectivity. A single amino acid, which is tyrosine in PPAR alpha and histidine in PPAR gamma, imparts subtype selectivity for both thiazolidinedione and nonthiazolidinedione ligands. The availability of high-resolution cocrystal structures of the three PPAR subtypes will aid the design of drugs for the treatments of metabolic and cardiovascular diseases.

Autoregulation of the human liver X receptor alpha promoter.

Previous work has implicated the nuclear receptors liver X receptor alpha (LXR alpha) and LXR beta in the regulation of macrophage gene expression in response to oxidized lipids. Macrophage lipid loading leads to ligand activation of LXRs and to induction of a pathway for cholesterol efflux involving the LXR target genes ABCA1 and apoE. We demonstrate here that autoregulation of the LXR alpha gene is an important component of this lipid-inducible efflux pathway in human macrophages. Oxidized low-density lipoprotein, oxysterols, and synthetic LXR ligands induce expression of LXR alpha mRNA in human monocyte-derived macrophages and human macrophage cell lines but not in murine peritoneal macrophages or cell lines. This is in contrast to peroxisome proliferator-activated receptor gamma (PPAR gamma)-specific ligands, which stimulate LXR alpha expression in both human and murine macrophages. We further demonstrate that LXR and PPAR gamma ligands cooperate to induce LXR alpha expression in human but not murine macrophages. Analysis of the human LXR alpha promoter led to the identification of multiple LXR response elements. Interestingly, the previously identified PPAR response element (PPRE) in the murine LXR alpha gene is not conserved in humans; however, a different PPRE is present in the human LXR 5'-flanking region. These results have implications for cholesterol metabolism in human macrophages and its potential to be regulated by synthetic LXR and/or PPAR gamma ligands. The ability of LXR alpha to regulate its own promoter is likely to be an integral part of the macrophage physiologic response to lipid loading.

Developmentally regulated cell cycle dependence of swelling-activated anion channel activity in the mouse embryo.

Anion channels activated by increased cell volume are a nearly ubiquitous mechanism of cell volume regulation, including in early preimplantation mouse embryos. Here, we show that the swelling-activated anion current (I(Cl,swell)) in early mouse embryos is cell-cycle dependent, and also that this dependence is developmentally regulated. I(Cl,swell) is present both in first meiotic prophase (germinal vesicle stage) mouse oocytes and in unfertilized mature oocytes in second meiotic metaphase, and it persists after fertilization though the 1-cell and 2-cell stages. I(Cl,swell) was found to remain unchanged during metaphase at the end of the 1-cell stage. However, I(Cl,swell) decreased during prophase and became nearly undetectable upon entry into metaphase at the end of the 2-cell stage. Entry into prophase/metaphase was required for the decrease in I(Cl,swell) at the end of the 2-cell stage, since it persisted indefinitely in 2-cell embryos arrested in late G(2). There is considerable evidence that the channel underlying I(Cl,swell) is not only permeable to inorganic anions, but to organic osmolytes as well. We found a similar pattern of cell cycle and developmental dependence in the 1-cell and 2-cell stages for the swelling-induced increase in permeability to the organic osmolyte glycine. Thus, entry into metaphase deactivates I(Cl,swell) in embryos, but only after developmental progression through the 2-cell stage.

Health issues in journalism and reporting.

Journalists witness events and report the news. The gathering and presentation of this information may subject the journalist to a variety of physical and psychological hazards. Some health and safety risk factors are inherent within the profession: for example, stress associated with deadlines and reporting on events in dangerous climactic or social conditions. Survey data and information collected by professional journalism associations should be used to educate journalists and, whenever possible, to attenuate the risk factors. Some of the more common risks within the profession include those relating to travel, repetitive strain, and psychological stress.

Liver X receptor (LXR) regulation of the LXRalpha gene in human macrophages.

The nuclear oxysterol receptors LXRalpha (NR1H3) and LXRbeta (NR1H2) coordinately regulate the expression of genes involved in the transport and catabolism of cholesterol. In macrophages, LXR stimulates the transcription of genes encoding transporters involved in cholesterol efflux, which may limit the transformation of these cells into foam cells in response to lipid loading. Here, we report that natural and synthetic LXR ligands induce the expression of the LXRalpha gene in primary human macrophages and differentiated THP-1 macrophages. This regulation was not observed in primary human adipocytes or hepatocytes, a human intestinal cell line, or in any mouse tissue or cell line examined. The human LXRalpha gene was isolated, and the transcription initiation site delineated. Analysis of the LXRalpha promoter revealed a functional LXR/RXR binding site approximately 2.9 kb upstream of the transcription initiation site. We conclude that LXRalpha regulates its own expression in human macrophages and that this response is likely to amplify the effects of oxysterols on reverse cholesterol transport. These findings underscore the importance of LXR as a potential therapeutic target for the treatment of atherosclerosis.

Catecholamines decrease nitric oxide production by cytokine-stimulated hepatocytes.

BACKGROUND: Catecholamines are significantly elevated in inflammatory responses and play a regulatory role in sepsis. Nitric oxide (NO), also a key inflammatory mediator in sepsis, is produced in large amounts by the inducible nitric oxide synthase (iNOS) in the liver. The purpose of this study was to test the hypothesis that catecholamines play a role in the regulation of NO production by hepatocytes. METHODS: Primary hepatocytes were isolated from healthy male Sprague-Dawley rats and either cultured with normal medium or stimulated with cytomix (interleukin-1 beta, interferon-gamma, and tumor necrosis factor-alpha) in the presence or absence of epinephrine or norepinephrine at varying concentrations. Total RNA was isolated 6 hours after treatment and analyzed by Northern blotting for iNOS mRNA. Protein extracts were obtained at 12 hours and were analyzed by Western immunoblotting for iNOS. Cell culture supernatants were analyzed for NO, determined as the stable end-product NO(2)(-), at 24 hours. RESULTS: Epinephrine and norepinephrine significantly decreased NO(2)(-) levels in stimulated hepatocytes but had no effect on iNOS mRNA or protein levels. The decrease in NO(2)(-) was reproduced by the adenylate cyclase stimulator, forskolin. The catecholamine-induced decrease in NO(2)(-) was completely reversed by the protein kinase A inhibitor Rp-8-Br-cyclic adenosine monophosphate. CONCLUSIONS: Catecholamines decrease hepatocyte production of NO in response to cytokine stimulation. This effect seems to be due to post-translational events and appears to be mediated in part by cyclic adenosine monophosphate.

Minimal residual disease (MRD) in remission t(8;21) AML and in vivo differentiation detected by FISH and CD34+ cell sorting.

Many patients with t(8;21) AML have residual positive cells during remission. We previously developed D-FISH probes that detect both derivative chromosomes and the normal alleles. In negative controls, only 2/44,000 (0.0045%) positive signals were observed. To investigate MRD, we examined specimens from 29 patients who had initially obtained CR. In remission patients, 61% had 1-4/2000 positive cells (0.05-0.19%). Higher frequencies were found in two patients in early relapse and in one patient in early remission. However, a negative test did not exclude relapse. Since false positives were negligible and because most t(8;21) AMLs express CD34, we asked whether cell sorting combined with FISH would increase the sensitivity. In one patient, we observed that 80% of CD34+ cells were t(8;21)+ at 2 months from initial clinical and cytogenetic remission. However, by 5 months the pre- and post-sorted populations contained 0.15% and 0.06% t(8;21) cells, respectively. Whereas essentially all t(8;21) cells in the initial specimen expressed CD34, only 0.6% were subsequently CD34+. These results are consistent with in vitro assays showing that residual t(8;21) cells undergo differentiation. Thus, FISH can identify MRD in a majority of t(8;21) patients and, combined with CD34+ selection, may provide an indirect assessment of the differentiation state of residual t(8;21) cells.

The human nuclear xenobiotic receptor PXR: structural determinants of directed promiscuity.

The human nuclear pregnane X receptor (hPXR) activates cytochrome P450-3A expression in response to a wide variety of xenobiotics and plays a critical role in mediating dangerous drug-drug interactions. We present the crystal structures of the ligand-binding domain of hPXR both alone and in complex with the cholesterol-lowering drug SR12813 at resolutions of 2.5 and 2.75 angstroms, respectively. The hydrophobic ligand-binding cavity of hPXR contains a small number of polar residues, permitting SR12813 to bind in three distinct orientations. The position and nature of these polar residues were found to be critical for establishing the precise pharmacologic activation profile of PXR. Our findings provide important insights into how hPXR detects xenobiotics and may prove useful in predicting and avoiding drug-drug interactions.

Recent trends in participation in physical education among US high school students.

To examine recent trends in physical education (PE) enrollment, daily attendance in PE, and being physically active in PE among high school students in the United States, this study analyzed data from the 1991, 1993, 1995, and 1997 national school-based Youth Risk Behavior Surveys (n = 55,734). Logistic regression analyses were conducted to test for significant linear time trends among the total student population and demographic subgroups (gender, race/ethnicity, and grade). Although PE enrollment in the total student population did not change from 1991 (48.9%) to 1997 (48.8%), the prevalence of students who attended PE daily, and the prevalence of students who were physically active > 20 minutes in an average PE class both decreased significantly among nearly all demographic subgroups. The prevalence of students who were physically active > 20 minutes in daily PE classes decreased from 34.2% in 1991 to 21.7% in 1997 (p < 0.001). To reverse current trends, high schools should implement daily PE classes that emphasize participation in lifelong health-related physical activity for all students.

Pharmacophore analysis of the nuclear oxysterol receptor LXRalpha.

A cell-free assay was developed for the orphan nuclear receptor LXRalpha that measures the ligand-dependent recruitment of a peptide from the steroid receptor coactivator 1 (SRC1) to the nuclear receptor. Using this ligand-sensing assay (LiSA), the structural requirements for activation of the receptor by oxysterols and related compounds were studied. The minimal pharmacophore for receptor activation was shown to be a sterol with a hydrogen bond acceptor at C24. 24(S),25-Epoxycholesterol (1), which meets this criterion, is among the most efficacious of the oxysterols and is an attractive candidate as the LXRalpha natural hormone. Cholenic acid dimethylamide (14) showed increased efficacy compared to 1, whereas the unnatural oxysterol 22(S)-hydroxycholesterol (4) was shown to be an antagonist of 1 in the LiSA. The structural requirements for SRC1 recruitment in the LiSA correlated with the transcriptional activity of compounds in a cell-based reporter assay employing LXRalpha-GAL4 chimeric receptors. Site-directed mutagenesis identified Trp(443) as an amino acid critical for activation of LXRalpha by oxysterol ligands. This information was combined with the structure-activity relationship developed from the LiSA to develop a 3D homology model of LXRalpha. This model may aid the design of synthetic drugs targeted at this transcriptional regulator of cholesterol homeostasis.

Simultaneous detection of multiple genetic aberrations in single cells by spectral fluorescence in situ hybridization.

Spectral fluorescence in situ hybridization (S-FISH) is a novel molecular cytogenetic approach that detects multiple disease-specific chromosomal aberrations in interphase nuclei using combinatorial fluorescence and digital imaging microscopy. A panel of six centromeric probes for chromosomes 7, 8, 9, 10, X, and Y, using a unique two-dye combination of four fluorophores, was developed to assess ploidy in breast tumors, bladder washings, and leukemia. Validation of S-FISH was performed by classic cytogenetics when metaphases were available or by standard fluorescence in situ hybridization (FISH) analyses. S-FISH identified clonal aberrations in newly diagnosed breast tumors and recurrent bladder cancer and revealed minimal residual disease in hyperdiploid acute lymphocytic leukemia, providing "proof of concept." Like standard FISH, aberrations were identified in poor growth/no growth specimen at the single cell level; however, S-FISH provided increased sensitivity over standard FISH by surveying six genetic targets instead of one or two. Disadvantages of the current assay include labor intensive screening and interpretative challenges with signal overlap in highly aneuploid samples and focal plane distortions. S-FISH appears to be a sensitive oncology assay with significant clinical application for early detection of new or reemerging clones, allowing for earlier therapeutic intervention and development of probe panels for individualized therapy.

Surveillance for characteristics of health education among secondary schools--school health education profiles, 1998.

PROBLEM/CONDITION: School health education (e.g., classroom instruction) is an essential component of school health programs; such education promotes the health of youth and improves overall public health. REPORTING PERIOD: February-May 1998. DESCRIPTION OF SYSTEM: The School Health Education Profiles monitor characteristics of health education in middle or junior high schools and senior high schools in the United States. The Profiles are school-based surveys conducted by state and local education agencies. This report summarizes results from 36 state surveys and 10 local surveys conducted among representative samples of school principals and lead health education teachers. The lead health education teacher coordinates health education policies and programs within a middle/junior high school or senior high school. RESULTS: During the study period, most schools in states and cities that conducted Profiles required health education in grades 6-12. Of these, a median of 91.0% of schools in states and 86.2% of schools in cities taught a separate health education course. The median percentage of schools in each state and city that tried to increase student knowledge in selected topics (i.e., prevention of tobacco use, alcohol and other drug use, pregnancy, human immunodeficiency virus [HIV] infection, other sexually transmitted diseases, violence, or suicide; dietary behaviors and nutrition; and physical activity and fitness) was >73% for each of these topics. The median percentage of schools with a health education teacher who coordinated health education was 38.7% across states and 37.6% across cities. A median of 41.8% of schools across states and a median of 31.0% of schools across cities had a lead health education teacher with professional preparation in health and physical education, whereas a median of 6.0% of schools across states and a median of 5.5% of schools across cities had a lead health education teacher with professional preparation in health education only. A median of 19.3% of schools across states and 21.2% of schools across cities had a school health advisory council. The median percentage of schools with a written school or school district policy on HIV-infected students or school staff members was 69.7% across states and 84.4% across cities. INTERPRETATION: Many middle/junior high schools and senior high schools require health education to help provide students with knowledge and skills needed for adoption of a healthy lifestyle. However, these schools might not be covering all important topic areas or skills sufficiently. The number of lead health education teachers who are academically prepared in health education and the number of schools with school health advisory councils needs to increase. PUBLIC HEALTH ACTION: The Profiles data are used by state and local education officials to improve school health education.

Use of birth control pills, condoms, and withdrawal among U.S. high school students.

PURPOSE: To examine the use of contraception at last sexual intercourse among currently sexually active adolescents. METHODS: We analyzed data from national school-based Youth Risk Behavior Surveys (YRBS) conducted in 1991, 1993, 1995, and 1997. The YRBS is a self-administered, anonymous survey which uses a national probability sample of U.S. students in public and private schools from grades 9 through 12. RESULTS: From 1991 to 1997, condom use significantly increased (from 46% to 57%), birth control pill use decreased (from 21% to 17%), and use of withdrawal significantly decreased (from 18% to 13%). In 1997, although more students were using condoms, 13% reported using withdrawal and 15% reported using no method to prevent pregnancy at last sexual intercourse. In 1997, condom use among females was significantly lower in the 9th grade than in the 12th grade (p <.001), whereas birth control pill use was higher (p <.001) and use of withdrawal remained stable. Among males, condom use and withdrawal use remained stable from 9th to 12th grade, whereas birth control pill use by their partner increased (p <.001). CONCLUSIONS: Inadequate contraceptive use among sexually active adolescents continues to be a major public health problem in the United States. For young people who will not remain sexually abstinent, families, health care providers, schools, and other influential societal institutions should promote the correct and continued use of condoms as essential protection against sexually transmitted diseases and human immunodeficiency virus infection.

Contextual influences on school provision of health services.

PURPOSE: To examine contextual factors that may facilitate or impede the provision of school health services. METHODS: Using a composite database derived primarily from the National Longitudinal Study of Adolescent Health, we used logistic regression to examine how selected characteristics of communities, schools, and state-level policies are related to the provision of specific health services by high schools. RESULTS: Schools whose students experienced more health risks were generally more likely to provide related services than schools whose students experienced fewer risks. State policies and requirements for health-related programs and services were associated with greater school-based provision of services. Availability of health care services within the community was associated with a reduced likelihood that schools provided similar services on-site; however, for some health services, the reverse was true. In general, more affluent communities were more likely to provide school health services than less affluent communities. Public schools were more likely to offer health services than private schools. CONCLUSIONS: Certain characteristics of communities, schools, and state-level policies are associated with the provision of school health services. These contextual factors appear to operate by creating a demand for services and by creating the opportunity for schools to provide health services.

Twenty-four-color spectral karyotyping reveals chromosome aberrations in cytogenetically normal acute myeloid leukemia.

Multicolor spectral karyotyping allows simultaneous visualization of all human chromosomes and screening for chromosomal rearrangements without a priori knowledge of any abnormalities involved. Based on this potentially increased sensitivity, we investigated, in a preliminary manner, whether spectral karyotyping could detect cytogenetic aberrations in karyotypically normal leukemia. The test population was comprised of 28 cryopreserved, cytogenetically normal acute myeloid leukemia (AML) samples from patients registered to a randomized trial for previously untreated AML (SWOG 9031). Two normal and 12 samples with known cytogenetic aberrations were used to validate and establish the diagnostic accuracy of the spectral karyotyping assay and instrumentation in a clinical setting. Enumeration and region-specific DNA fluorescence in situ hybridization (FISH) probes verified discrepant results. In the validation data set, spectral karyotyping refined complex karyotypic rearrangements in six cases and defined the chromosomal origin of a "jumping" homogeneously staining region; however, the technology was less sensitive in the detection of subtelomeric rearrangements and double minute chromosomes. In the test population, spectral karyotyping identified previously undetected cytogenetic aberrations in two cases (7%) of karyotypically normal AML: a cryptic 11q23 translocation in 20/20 cells and a minor monosomy 7 clone in 3/21 cells (FISH, 10.5%). Both of these abnormalities are considered to confer a poor prognosis when based on classical cytogenetic prognostic criteria. As an adjunct to classical cytogenetics and standard FISH analyses, the additive resolution of spectral karyotyping, in particular, with chromosome paints spiked with subtelomeric and/or locus-specific probes, may allow significant gains to be made in diagnostic accuracy and recognition of genotype/phenotype prognostic relationships, and in defining underlying biologic mechanisms in cancer. Genes Chromosomes Cancer 28:318-328, 2000.

St. John's wort induces hepatic drug metabolism through activation of the pregnane X receptor.

St. John's wort (Hypericum perforatum) is an herbal remedy used widely for the treatment of depression. Recent clinical studies demonstrate that hypericum extracts increase the metabolism of various drugs, including combined oral contraceptives, cyclosporin, and indinavir. In this report, we show that hyperforin, a constituent of St. John's wort with antidepressant activity, is a potent ligand (K(i) = 27 nM) for the pregnane X receptor, an orphan nuclear receptor that regulates expression of the cytochrome P450 (CYP) 3A4 monooxygenase. Treatment of primary human hepatocytes with hypericum extracts or hyperforin results in a marked induction of CYP3A4 expression. Because CYP3A4 is involved in the oxidative metabolism of >50% of all drugs, our findings provide a molecular mechanism for the interaction of St. John's wort with drugs and suggest that hypericum extracts are likely to interact with many more drugs than previously had been realized.

Physical activity, food choice, and weight management goals and practices among US college students.

INTRODUCTION: Physical activity and a healthy diet have been recommended to help reverse the increasing prevalence of overweight among adolescents and adults in the United States. METHODS: Data is from the 1995 National College Health Risk Behavior Survey. A representative sample of US undergraduate college students (n = 4609) were analyzed to examine associations of physical activity and food choice with weight management goals and practices. RESULTS: Based on self-reported height and weight, 35% of students were overweight or obese (body mass index > or = 25.0). Nearly half (46%) of all students reported they were trying to lose weight. Female students were less likely than male students to be overweight, but more likely to be trying to lose weight. Among female and male students, using logistic regression to control for demographics, trying to lose weight was associated with participation in vigorous physical activity and strengthening exercises, and consumption of < or = 2 servings/ day of high-fat foods. Female and male students who reported using exercise to lose weight or to keep from gaining weight were more likely than those who did not to participate in vigorous, strengthening, and moderate physical activity, and were more likely to eat > or = 5 servings/day of fruits and vegetables and < or = 2 servings/day of high-fat foods. Among students who were trying to lose weight, only 54% of females and 41% of males used both exercise and diet for weight control. CONCLUSION: Colleges should implement programs to increase student awareness of healthy weight management methods and the importance of physical activity combined with a healthy diet.

Orphan nuclear receptors constitutive androstane receptor and pregnane X receptor share xenobiotic and steroid ligands.

Xenobiotics induce the transcription of cytochromes P450 (CYPs) 2B and 3A through the constitutive androstane receptor (CAR; NR1I3) and pregnane X receptor (PXR; NR1I2), respectively. In this report, we have systematically compared a series of xenobiotics and natural steroids for their effects on mouse and human CAR and PXR. Our results demonstrate dual regulation of PXR and CAR by a subset of compounds that affect CYP expression. Moreover, there are marked pharmacological differences between the mouse (m) and human (h) orthologs of both CAR and PXR. For example, the planar hydrocarbon 1, 4-bis[2-(3,5-dichloropyridyl-oxy)]benzene activates mCAR and hPXR but has little or no activity on hCAR and mPXR. In contrast, the CAR deactivator androstanol activates both mouse and human PXR. Similarly, the PXR activator clotrimazole is a potent deactivator of hCAR. Using radioligand binding and fluorescence resonance energy transfer assays, we demonstrate that several of the compounds that regulate mouse and human CAR, including natural steroids, bind directly to the receptors. Our results suggest that CAR, like PXR, is a steroid receptor that is capable of recognizing structurally diverse compounds. Moreover, our findings underscore the complexity in the physiologic response to xenobiotics.