Impact of muffle furnace preparation on the results of crystalline silica analysis.

A prior report demonstrated an unacceptably low level of accuracy in silica analytical testing, with a general negative bias (i.e., underreporting) although other inaccuracies included false-positive results when analyzing blank filters. The possible bias may have been due to the loss of sample during shipping and or sample preparation. We report on a follow-up study that was designed to mimic the original study, but in which sources of variability were evaluated. We found no effect on silica recoveries due to shipping and confirmed the prior study results that the muffle furnace ashing process led to low overall recoveries (49-104%), depending on the adherence to the recommended preparation method. Plasma ashing recoveries ranged from 89 to 108%. Our results suggest that muffle-furnace ashing using a crucible should be restricted. More broadly, however, muffle-furnace ashing is only one source of analytical error that contributes to the relatively poor overall performance revealed by Cox et al. Whatever the case, OSHA should ensure that its proposed requirements to improve laboratory performance will actually lead to the discovery and correction of all major sources of error by participating laboratories. This is particularly important in light of OSHA's proposed reduction in the PEL and action level proposed by OSHA.

Monoclonal antibody against macrophage colony-stimulating factor suppresses circulating monocytes and tissue macrophage function but does not alter cell infiltration/activation in cutaneous lesions or clinical outcomes in patients with cutaneous lupus erythematosus.

This study's objective was to assess the effects of PD-0360324, a fully human immunoglobulin G2 monoclonal antibody against macrophage colony-stimulating factor in cutaneous lupus erythematosus (CLE). Patients with active subacute CLE or discoid lupus erythematosus were randomized to receive 100 or 150 mg PD-0360324 or placebo via intravenous infusion every 2 weeks for 3 months. Blood and urine samples were obtained pre- and post-treatment to analyse pharmacokinetics and pharmacodynamic changes in CD14(+) CD16(+) monocytes, urinary N-terminal telopeptide (uNTX), alanine/aspartate aminotransferases (ALT/AST) and creatine kinase (CK); tissue biopsy samples were taken to evaluate macrophage populations and T cells using immunohistochemistry. Clinical efficacy assessments included the Cutaneous Lupus Erythematosus Disease Area and Severity Index (CLASI). Among 28 randomized/analysed patients, peak/trough plasma concentrations increased in a greater-than-dose-proportional manner with dose increases from 100 to 150 mg. Statistically significant differences were observed between active treatment and placebo groups in changes from baseline in CD14(+) CD16(+) cells, uNTX, ALT, AST and CK levels at most time-points. The numbers, density and activation states of tissue macrophages and T cells did not change from baseline to treatment end. No between-group differences were seen in CLASI. Patients receiving PD-0360324 reported significantly more adverse events than those receiving placebo, but no serious adverse events. In patients with CLE, 100 and 150 mg PD-0360324 every 2 weeks for 3 months suppressed a subset of circulating monocytes and altered activity of some tissue macrophages without affecting cell populations in CLE skin lesions or improving clinical end-points.

How reliable are crystalline silica dust concentration measurements?

To determine how reliably commercial laboratories measure crystalline silica concentrations corresponding to OSHA's proposed limits, 105 filters were prepared with known masses of 20, 40, and 80 µg of respirable quartz corresponding to airborne silica concentrations of 25, 50, and 100 µg/m(3) and were submitted, in a blind test, to qualified commercial laboratories over a nine month period. Under these test conditions, the reported results indicated a lack of accuracy and precision needed to reliably inform regulatory compliance decisions. This was true even for filters containing only silica, without an interfering matrix. For 36 filters loaded with 20 or more micrograms of silica, the laboratories reported non-detected levels of silica. Inter-laboratory variability in this performance test program was so high that the reported results could not be used to reliably discriminate among filters prepared to reflect 8-h exposures to respirable quartz concentrations of 25, 50 and 100 µg/m(3). Moreover, even in intra-laboratory performance, there was so much variability in the reported results that 2-fold variations in exposure concentrations could not be reliably distinguished. Part of the variability and underreporting may result from the sample preparation process. The results of this study suggest that current laboratory methods and practices cannot necessarily be depended on, with high confidence, to support proposed regulatory standards with reliable data.

Genetical genomics of Th1 and Th2 immune response in a baboon model of atherosclerosis risk factors.

OBJECTIVE: CD4(+) T-cells mediate inflammation in atherosclerosis, but additive genetic effects on associated pathways of Th1 and Th2 immune response have not been described. We sought to characterize heritability, pleiotropy, and QTL effects on the expression of genes implicated in Th1 and Th2 immune response in a baboon model of risk factors for atherosclerosis. METHODS: We employed a maximum likelihood-based variance decomposition approach to estimate additive genetic effects on transcript levels generated from a gene expression profile of lymphocytes in 499 pedigreed baboons maintained on a basal diet. Transcript levels for 57 genes implicated in Th1 and Th2 immune response were selected for analysis based on significant heritability in this profile. Multipoint whole genome scans were conducted on heritable transcript levels to localize QTLs influencing these measures. To evaluate pleiotropic effects on transcript levels, we estimated genetic and phenotypic correlations among transcript measures, and assessed their correspondence using a Mantel test. Network analysis using GeneGo's MetaCore™ software was conducted to characterize known interaction among coded proteins. RESULTS: Heritabilities for candidate gene transcript levels ranged from 0.092-0.786 (median h(2)=0.278, P=4.72×10(-4)). Linkage analyses yielded significant evidence (LOD≥2.73) for 14 eQTLs (LOD score range 2.76-14.87, genome-wide P=4.9×10(-2)-1.03×10(-14)). Estimates of genetic correlation supported shared additive genetic effects incorporating all 57 transcripts (null hypothesis of ρ(G)=0 rejected at FDR≤0.05 for 522 of 1596 estimates), and accounted for most of the observed phenotypic correlation among transcripts (Mantel test, r([ρP],)([ρG])=0.781, P<0.0001). Network analysis revealed direct interactions among 54 of the 57 coded proteins. CONCLUSIONS: We conclude that major genetic effects influence expression levels of multiple genes implicated in Th1 and Th2 immune response. Additionally, we find that expression levels of these candidate genes are characterized by extensive pleiotropy, consistent with known interaction among their coded proteins, many of which are independently associated with atherosclerosis.

Genetics of variation in adiponectin in pedigreed baboons: evidence for pleiotropic effects on adipocyte volume and serum adiponectin.

To detect and localize the effects of genes influencing variation in adiponectin mRNA and protein levels, we conducted statistical genetic analyses of circulating concentrations of adiponectin and adiponectin (ADIPOQ) mRNA expression in omental adipose tissue in adult, pedigreed baboons (Papio anubis). An omental adipose tissue biopsy and blood sample were collected from 427 baboons from the colony at the Southwest Foundation for Biomedical Research, San Antonio, TX. Total RNA was isolated from adipose tissue and adiponectin mRNA levels were assayed by real-time, quantitative reverse transcriptase-PCR. Adiponectin, insulin, glucose, cholesterol, high-density lipoproteins and triglycerides were measured in fasting serum. Quantitative genetic analyses were conducted for adiponectin mRNA and serum protein using a maximum likelihood-based variance decomposition approach. A genome-wide linkage analysis was conducted using adiponectin mRNA and protein levels as phenotypes. Significant heritability was estimated for ADIPOQ mRNA levels (h2=0.19+/-0.07, P=0.01) and protein levels (h2=0.28+/-0.14, P=0.003). Genetic correlations were found between adiponectin protein and body weight (rho(G)=-0.51, P=0.03), cell volume (rho(G)=-0.73, P=0.04), serum triglycerides (rho(G)=-0.67, P=0.03), and between adiponectin mRNA and glucose (rho(G)=0.93, P<0.01). A logarithm of odds score of 2.9 was found for ADIPOQ mRNA levels on baboon chromosome 4p, which is orthologous to human 6p21. There is a significant genetic component affecting variation in the analyzed traits, and common genes may be influencing adiponectin expression, adipocyte volume, body weight and circulating triglycerides. The region on 6p21 has been linked to diabetes-related phenotypes in human studies.

A pleiotropic QTL on 2p influences serum Lp-PLA2 activity and LDL cholesterol concentration in a baboon model for the genetics of atherosclerosis risk factors.

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), the major portion of which is bound to low-density lipoprotein, is an independent biomarker of cardiovascular disease risk. To search for common genetic determinants of variation in both Lp-PLA(2) activity and LDL cholesterol (LDL-C) concentration, we assayed these substances in serum from 679 pedigreed baboons. Using a maximum likelihood-based variance components approach, we detected significant evidence for a QTL affecting Lp-PLA(2) activity (LOD=2.79, genome-wide P=0.039) and suggestive evidence for a QTL affecting LDL-C levels (LOD=2.16) at the same location on the baboon ortholog of human chromosome 2p. Because we also found a significant genetic correlation between the two traits (rho(G)=0.50, P<0.00001), we conducted bivariate linkage analyses of Lp-PLA(2) activity and LDL-C concentration. These bivariate analyses improved the evidence (LOD=3.19, genome-wide P=0.015) for a QTL at the same location on 2p, corresponding to the human cytogenetic region 2p24.3-p23.2. The QTL-specific correlation between the traits (rho(Q)=0.62) was significantly different from both zero and 1 (P[rho(Q)=0]=0.047; P[rho(Q)=1]=0.022), rejecting the hypothesis of co-incident linkage and consistent with incomplete pleiotropy at this locus. We conclude that polymorphisms at the QTL described in this study exert some genetic effects that are shared between Lp-PLA(2) activity and LDL-C concentration.

Effect of 30 per cent maternal nutrient restriction from 0.16 to 0.5 gestation on fetal baboon kidney gene expression.

Previous studies in rodents and sheep show that maternal nutrient restriction during pregnancy alters fetal renal development. To date, no studies using fetal baboon RNA with human Affymetrix gene chips have been published. In the present study we have (1) evaluated the specificity of the Affymetrix human gene array 'Laboratory on a Chip' system for use with fetal baboon mRNA and (2) investigated the effects of moderate maternal global nutrient restriction (NR; 70% of ad libitum animals) from early (30 days gestation (dG)) to mid-gestation (90 dG; term = 184 dG) on the fetal baboon kidney. Morphometric and blood measurements were made on 12 non-pregnant baboons before they were bred. All baboons were fed ad libitum until 30 days pregnant, at which time six control baboons continued to feed ad libitum (control - C) while six received 70% of the C diet on a weight adjusted basis. Fetal kidneys were collected following caesarean section at 90 dG, with samples flash frozen and fixed for histological assessment. Fetal hip circumference was decreased in the NR group (68 +/- 2 versus 75 +/- 2 mm), while fetal body weight and all other measurements of fetal size were not different between C and NR at 90 dG. Maternal body weight was decreased in the NR group (12.16 +/- 0.34 versus 13.73 +/- 0.55 kg). Having established the specificity of the Affymetrix system for fetal baboon mRNA, gene expression profiling of fetal kidneys in the context of our maternal nutrient restriction protocol shows that NR resulted in a down-regulation of genes in pathways related to RNA, DNA and protein biosynthesis, metabolism and catabolism. In contrast, genes in cell signal transduction, communication and transport pathways were up-regulated in the NR group. These changes indicate that even a moderate level of maternal global NR impacts fetal renal gene pathways. Our histological assessment of renal structure indicates decreased tubule density within the cortex of NR kidneys compared with controls. The number of glomerular cross-sections per unit area were unaffected by NR, suggesting that tubule tortuosity and/or tubule length was decreased in the NR kidney. Taken together the changes indicate that NR results in accelerated fetal renal differentiation. The negative impact of poor maternal nutrition on the fetal kidney may therefore be in part due to shortening of critical phases of renal growth resulting in decreased functional capacity in later life. These findings may have important implications for postnatal renal function, thereby contributing to the observed increased predisposition to hypertension and renal disease in the offspring of nutrient restricted mothers.

Science-policy in environmental and health risk assessment: if we cannot do without, can we do better?

How can empirical evidence of adverse effects from exposure to noxious agents, which is often incomplete and uncertain, be used most appropriately to protect human health? We examine several important questions on the best uses of empirical evidence in regulatory risk management decision-making raised by the US Environmental Protection Agency (EPA)'s science-policy concerning uncertainty and variability in human health risk assessment. In our view, the US EPA (and other agencies that have adopted similar views of risk management) can often improve decision-making by decreasing reliance on default values and assumptions, particularly when causation is uncertain. This can be achieved by more fully exploiting decision-theoretic methods and criteria that explicitly account for uncertain, possibly conflicting scientific beliefs and that can be fully studied by advocates and adversaries of a policy choice, in administrative decision-making involving risk assessment. The substitution of decision-theoretic frameworks for default assumption-driven policies also allows stakeholder attitudes toward risk to be incorporated into policy debates, so that the public and risk managers can more explicitly identify the roles of risk-aversion or other attitudes toward risk and uncertainty in policy recommendations. Decision theory provides a sound scientific way explicitly to account for new knowledge and its effects on eventual policy choices. Although these improvements can complicate regulatory analyses, simplifying default assumptions can create substantial costs to society and can prematurely cut off consideration of new scientific insights (e.g., possible beneficial health effects from exposure to sufficiently low 'hormetic' doses of some agents). In many cases, the administrative burden of applying decision-analytic methods is likely to be more than offset by improved effectiveness of regulations in achieving desired goals. Because many foreign jurisdictions adopt US EPA reasoning and methods of risk analysis, it may be especially valuable to incorporate decision-theoretic principles that transcend local differences among jurisdictions.

Cross-species replication of a serum osteocalcin quantitative trait locus on human chromosome 16q in pedigreed baboons.

Osteocalcin (OC), a serum marker of bone formation, in its intact form reflects osteoblast activity. It is of interest to clinicians and bone biologists due to easy measurability and potential utility as an identifier of those at risk for fracture and other complications associated with bone metabolism disorders. The only published linkage study in humans shows significant evidence for a quantitative trait locus (QTL) affecting OC levels on 16q. We used the baboon, a primate model for skeletal maintenance and turnover, to detect and quantify the effects of genes on serum OC levels and to localize chromosomal regions harboring the responsible loci. We assayed OC levels in 591 pedigreed animals, assessed OC heritability, and conducted a genomewide linkage scan for evidence of QTLs affecting this phenotype. Heritability in these baboons is 0.24. Suggestive linkage is evident with markers in a region homologous to human chromosome 16q. This first genomewide linkage scan in a nonhuman primate for QTLs affecting bone formation as reflected by OC levels provides cross-species replication of the QTL on chromosome 16q previously localized in humans. Given the concordance of results of the only two genome scans for this trait in two primate species, further studies of this region are warranted.

A quantitative trait locus for normal variation in forearm bone mineral density in pedigreed baboons maps to the ortholog of human chromosome 11q.

Baboons share many anatomical, physiological, and developmental characteristics with humans that make them excellent models for human bone maintenance and turnover. We conducted statistical genetic analyses, including a whole-genome linkage screen, of dual-energy x-ray absorptiometry-acquired measures of areal bone mineral density (aBMD), currently the most reliable single predictor of susceptibility to osteoporotic fracture in humans, from three forearm sites on the radius and ulna of 667 pedigreed baboons. We used a maximum likelihood-based variance decomposition approach to detect and quantify the effects of genes on normal variation in aBMD in the forearm of these baboons and to localize these effects to chromosomal regions. We estimated significant heritability for aBMD at all three sites and found evidence for a quantitative trait locus (QTL) contributing significantly to the genetic effects on this trait in a region of the baboon genome homologous to human chromosome 11q12-13. This first reported genome-wide linkage screen in a nonhuman primate for QTLs affecting forearm aBMD provides important cross-species replication of a QTL found in humans. The concordance of our results in a nonhuman primate with those reported for humans provides strong evidence that a gene (or genes) in this region affects normal variation in BMD.

Causation in risk assessment and management: models, inference, biases, and a microbial risk-benefit case study.

Causal inference of exposure-response relations from data is a challenging aspect of risk assessment with important implications for public and private risk management. Such inference, which is fundamentally empirical and based on exposure (or dose)-response models, seldom arises from a single set of data; rather, it requires integrating heterogeneous information from diverse sources and disciplines including epidemiology, toxicology, and cell and molecular biology. The causal aspects we discuss focus on these three aspects: drawing sound inferences about causal relations from one or more observational studies; addressing and resolving biases that can affect a single multivariate empirical exposure-response study; and applying the results from these considerations to the microbiological risk management of human health risks and benefits of a ban on antibiotic use in animals, in the context of banning enrofloxacin or macrolides, antibiotics used against bacterial illnesses in poultry, and the effects of such bans on changing the risk of human food-borne campylobacteriosis infections. The purposes of this paper are to describe novel causal methods for assessing empirical causation and inference; exemplify how to deal with biases that routinely arise in multivariate exposure- or dose-response modeling; and provide a simplified discussion of a case study of causal inference using microbial risk analysis as an example. The case study supports the conclusion that the human health benefits from a ban are unlikely to be greater than the excess human health risks that it could create, even when accounting for uncertainty. We conclude that quantitative causal analysis of risks is a preferable to qualitative assessments because it does not involve unjustified loss of information and is sound under the inferential use of risk results by management.

Genome-wide scan of resistin mRNA expression in omental adipose tissue of baboons.

INTRODUCTION: The hormone resistin was recently discovered in adipose tissue of mice. Functional tests suggest a role for resistin in the regulation of insulin sensitivity. However, human studies have reported controversial results on the metabolic function of this hormone. METHODS: A 1 g omental adipose tissue biopsy was obtained from 404 adult baboons. Resistin mRNA expression was assayed by real-time, quantitative RT-PCR, and univariate and bivariate quantitative genetic analyses were performed, via the variance decomposition approach. A genome scan analysis was conducted using resistin mRNA abundance in omental adipose tissue as a quantitative phenotype. RESULTS: A significant heritability of h2 = 0.23 (P = 0.003) was found for resistin mRNA abundance in omental adipose tissue. A genome scan detected a quantitative trait locus for resistin expression with an LOD score of 3.8, in the region between markers D19S431 and D19S714, corresponding to human chromosome 19 p13. This chromosomal region contains genes related to insulin resistance phenotypes, such as resistin, insulin receptor, angiopoietin-like 4 protein and LDL receptor. CONCLUSIONS: Individual variation in resistin mRNA expression has a significant genetic component, and a gene or genes on chromosome 19 p13 may regulate resistin mRNA levels in baboon omental adipose tissue.

Sodium-lithium countertransport activity is linked to chromosome 5 in baboons.

The genes involved in the regulation of cellular sodium transport characteristics, which are correlated with some forms of essential hypertension, have not yet been identified. We are studying the genes and environmental factors that affect red blood cell sodium-lithium countertransport (SLC) activity and intracellular sodium (ICNa) concentration in 634 baboons that comprise 11 pedigrees of 2 and 3 generations each. To detect and locate possible quantitative trait loci (QTLs) that affect SLC activity and ICNa concentration, we performed a genome screen by using a maximum likelihood-based variance-components linkage analysis program (SOLAR). SLC and ICNa phenotypes as well as genotypes on 281 microsatellite loci were available for all pedigreed animals. Both SLC and ICNa traits were highly heritable (residual heritability 0.593+/-0.083 [P<0.0001] and 0.739+/-0.082 [P<0.0001], respectively). We obtained evidence that a possible QTL for SLC activity is located on the baboon homologue of human chromosome 4 between D4S2456 and D4S2365 with a maximum multipoint lod score of 9.3 (P<10(-)(10)) near D4S1645. This QTL accounts for approximately two thirds of the total additive genetic variation in SLC activity in baboons. Although ICNa concentration was highly heritable, we found no evidence for linkage to a QTL with use of this methodology. Thus, we have evidence that a gene located on the baboon homologue of human chromosome 4 (baboon chromosome 5) affects cell sodium transport in baboons.

Sonographic assessment of hepatic transplants.

Liver transplantation has become a standard treatment for end-stage liver disease, with survival rates for liver transplant patients increasing steadily. Several imaging techniques are used to evaluate patients for possible complications of liver transplantation. This article focuses on the role of ultrasound and color flow and spectral Doppler in evaluating complications such as fluid collections and vascular or biliary obstructions.

Ultrasound and twin-twin transfusion syndrome.

This article examines twin-twin transfusion syndrome, a condition in which identical twins share a single placenta with abnormal anastomoses. The syndrome causes unequal blood flow between the twins that, if untreated, is likely to result in the death of one or both. Ultrasound is used to diagnose the syndrome and guide treatment, which may consist of aggressive amniocentesis, endoscopic laser surgery or selective feticide.