Skin sensitization to fragrance hydroperoxides: interplay between dendritic cells, keratinocytes and free radicals.

BACKGROUND: Skin sensitization to hydroperoxides (R-OOHs) of the commonly used fragrance terpenes limonene, linalool and citronellol is frequently reported. R-OOHs are believed to initiate the process leading to sensitization and allergic contact dermatitis through mechanisms involving radical intermediates. Thus, radical intermediates, keratinocytes and dendritic cells (DCs) may act in concert to initiate the process. OBJECTIVES: To evaluate individual DC activation profiles by R-OOHs in the context of keratinocytes with regard to frequency, specificity and magnitude of upregulation. METHODS: We used 2D and 3D cocultures with keratinocytes/reconstructed human epidermis (RHE) and DCs to evaluate cell surface levels of the costimulatory molecules CD86, CD80 and the adhesion molecule CD54 on cocultured DCs. Analysis of radical formation from limonene hydroperoxides in RHE was performed using electron paramagnetic resonance combined with the spin trapping technique. RESULTS: R-OOHs induce donor-dependent DC activation. Major differences were found between the limonene-OOHs. Limonene-1-OOH was stronger with respect to both frequency and magnitude of response. Using a 3D coculture model, no DC activation was detected after topical application of 0·2% limonene-OOHs (20 µg cm-2 ), while 1·2% limonene-1-OOH or 2% limonene-2-OOH induced DC activation. Furthermore, we demonstrated differences in the carbon and oxygen radicals formed from the limonene-OOHs using RHE, mimicking what may happen in vivo. CONCLUSIONS: We report clear individual differences in DC maturation induced by the most important hydroperoxides. Response rates and magnitude of response both indicate that very small structural alterations in the hydroperoxides are translated into specific DC responses. In addition, we provide more insight into the amounts of hydroperoxides that can activate DCs and induce sensitization.

Measurement of Strain in Cardiac Myocytes at Micrometer Scale Based on Rapid Scanning Confocal Microscopy and Non-Rigid Image Registration.

Measurement of cell shortening is an important technique for assessment of physiology and pathophysiology of cardiac myocytes. Many types of heart disease are associated with decreased myocyte shortening, which is commonly caused by structural and functional remodeling. Here, we present a new approach for local measurement of 2-dimensional strain within cells at high spatial resolution. The approach applies non-rigid image registration to quantify local displacements and Cauchy strain in images of cells undergoing contraction. We extensively evaluated the approach using synthetic cell images and image sequences from rapid scanning confocal microscopy of fluorescently labeled isolated myocytes from the left ventricle of normal and diseased canine heart. Application of the approach yielded a comprehensive description of cellular strain including novel measurements of transverse strain and spatial heterogeneity of strain. Quantitative comparison with manual measurements of strain in image sequences indicated reliability of the developed approach. We suggest that the developed approach provides researchers with a novel tool to investigate contractility of cardiac myocytes at subcellular scale. In contrast to previously introduced methods for measuring cell shorting, the developed approach provides comprehensive information on the spatio-temporal distribution of 2-dimensional strain at micrometer scale.

Wrong-site nerve blocks: 10 yr experience in a large multihospital health-care system.

INTRODUCTION: Although wrong-site surgery has garnered extensive scrutiny, the incidence of wrong-site blocks remains unknown. Our study thus sought to quantify the incidence of wrong-site blocks and examine some of their associated risk factors in our multihospital health-care system. METHODS: Using quality-improvement and billing data, we quantified the total number of blocks and wrong-site blocks occurring between July 1, 2002 and June 30, 2012 within the University of Pittsburgh Medical Center Health System. The incidence of wrong-site block was determined by block type, hospital, and type of service involved in performing the block. The incidence of wrong-site block was compared with that of wrong-site surgery. Fisher's exact tests were performed to determine associations between the incidence of wrong-site block and any of the aforementioned variables. A root-cause analysis was performed to determine the source of wrong-site blocks after the implementation of a timeout policy. RESULTS: Of the 85 915 patients receiving blocks, 70 441 received only unilateral blocks, yielding an overall incidence of wrong-site block of 1.28 (95% confidence interval 0.43-2.13) per 10 000 patients receiving unilateral blocks. The incidence of wrong-site block was highest with femoral blocks, and differed from the incidence of wrong-site surgery. All occurrences of wrong-site block after the implementation of the timeout policy involved policy violations. CONCLUSIONS: Our study provides the first incidence data on wrong-site block in a large patient population and can help hospitals to develop policies based on these data. It is yet to be determined whether active intervention can eliminate this adverse event.

Limited carbon storage in soil and litter of experimental forest plots under increased atmospheric CO2.

The current rise in atmospheric CO2 concentration is thought to be mitigated in part by carbon sequestration within forest ecosystems, where carbon can be stored in vegetation or soils. The storage of carbon in soils is determined by the fraction that is sequestered in persistent organic materials, such as humus. In experimental forest plots of loblolly pine (Pinus taeda) exposed to high CO2 concentrations, nearly half of the carbon uptake is allocated to short-lived tissues, largely foliage. These tissues fall to the ground and decompose, normally contributing only a small portion of their carbon content to refractory soil humic materials. Such findings call into question the role of soils as long-term carbon sinks, and show the need for a better understanding of carbon cycling in forest soils. Here we report a significant accumulation of carbon in the litter layer of experimental forest plots after three years of growth at increased CO2 concentrations (565 microl l(-1)). But fast turnover times of organic carbon in the litter layer (of about three years) appear to constrain the potential size of this carbon sink. Given the observation that carbon accumulation in the deeper mineral soil layers was absent, we suggest that significant, long-term net carbon sequestration in forest soils is unlikely.

Shade selection. Communicating with the laboratory technician.

Shade selection for anterior crowns has always set up a communications problem between the dentist and laboratory technician. Over the years, many different techniques have been formulated to help overcome the problem. These techniques include picture taking, drawing diagrams and using multiple porcelain shade guides. However, they have not completely erased the difficulty of communicating the choice of the proper shade of an anterior crown. This was especially true in the 1990's when all-ceramic crowns were introduced. Popular techniques dentists use for communicating shade selections will be reviewed, along with guidelines for making the proper selection. Many dentists are familiar only with the techniques they were taught in dental school and/or residency program and are unaware of the superior methods that can be used. This type of review can be extremely helpful to restorative dentists.

The study of candidate genes in drug trials: sample size considerations.

With discovery of an increasing number of candidate genes that may affect inter-individual variability in response to drugs, the design of drug trials that incorporate their study has become relevant. We discuss the determination of sample size for such studies when the number of tests to perform is given, or, alternatively, the number of tests to perform when the sample size is given. In many cases, a uniformly most powerful test does not exist and normal approximations are not sufficiently accurate to determine sample size. We discuss briefly various tests of interest and we give simple examples to illustrate some of the problems that arise.

What's behind your smile?

Dentistry in the 1990s has seen an evolution in new cosmetic materials and techniques. In order to appreciate and use these methods and materials properly, it is important to have an understanding of cosmetic dentistry and its origins. This article gives a brief history of esthetic dentistry and guidelines for cosmetic dentists of the future.

Interpreting recruitment limitation in forests.

Studies of tree recruitment are many, but they provide few general insights into the role of recruitment limitation for population dynamics. That role depends on the vital rates (transitions) from seed production to sapling stages and on overall population growth. To determine the state of our understanding of recruitment limitation we examined how well we can estimate parameters corresponding to these vital rates. Our two-part analysis consists of (1) a survey of published literature to determine the spatial and temporal scale of sampling that is basis for parameter estimates, and (2) an analysis of extensive data sets to evaluate sampling intensity found in the literature. We find that published studies focus on fine spatial scales, emphasizing large numbers of small samples within a single stand, and tend not to sample multiple stands or variability across landscapes. Where multiple stands are sampled, sampling is often inconsistent. Sampling of seed rain, seed banks, and seedlings typically span <1 yr and rarely last 5 yr. Most studies of seeding establishment and growth consider effects of a single variable and a single life history stage. By examining how parameter estimates are affected by the spatial and temporal extent of sampling we find that few published studies are sufficiently extensive to capture the variability in recruitment stages. Early recruitment stages are especially variable and require samples across multiple years and multiple stands. Ironically, the longest duration data sets are used to estimate mortality rates, which are less variable (in time) than are early life history stages. Because variables that affect recruitment rates interact, studies of these interactions are needed to assess their full impacts. We conclude that greater attention to spatially extensive and longer duration sampling for early life history stages is needed to assess the role of recruitment limitation in forests.

The impact of pharmacogenetics on the future of healthcare.

Pharmacogenetics has been promoted as potentially providing benefits to patients, managed care organizations and pharmaceutical companies. This has not translated into products that benefit healthcare developers, providers or consumers. The reasons for this are many, but this will change as the financial incentives become clear for the pharmaceutical industry to develop products that use genetic susceptibility as part of the rationale for products, healthcare providers have increasing incentive to reduce costs, and patients demand up-to-date technologies to optimize healthcare. Recent studies have established genetic contributions that alter the response to therapy for some disease entities, and more will follow as pharmacogenetics becomes increasingly accepted as an important consideration in the therapeutic decision-making process.

Isolation and genomic structure of a human homolog of the yeast periodic tryptophan protein 2 (PWP2) gene mapping to 21q22.3.

As part of efforts to identify candidate genes for disease mapping to the 21q22.3 region, we have assembled a 770-kb cosmid and BAC contig containing eight tightly linked markers. These cosmids and BACs were restriction mapped using eight rare cutting enzymes, with the goal of identifying CpG-rich islands. One such island was identified by the clustering of NotI, EagI, SstII, and BssHII sites, and corresponded to the NotI linking clone LJ104 described previously. A 7.6-kb HindIII fragment containing this CpG-rich island was subcloned and partially sequenced. A homology search using the sequence obtained from either side of the NotI site identified an expressed sequence tag with homology to the yeast periodic tryptophan protein 2 (PWP2). Several cDNAs corresponding to the human PWP2 gene were identified and partially sequenced. Northern blot analysis revealed a 3.3-kb transcript that was well expressed in all tissues tested. A cDNA consensus of 3157 bp was obtained, and an open reading frame potentially encoding 919 amino acid residues was identified. The predicted protein shows 42% identity and 57% similarity at the amino acid level to the yeast PWP2 protein, which is a member of the WD-repeat containing superfamily, and potentially encodes a G-protein beta subunit. The PWP2 gene is split into 21 exons, ranging in size from 53 to 516 bp, and spans an estimated 25 kb. The gene is transcribed in a 21cen-->21qter direction, with its 5' end mapping approximately 195 kb proximal to the 5' end of the phosphofructokinase-liver isoform gene. Four single base-pair polymorphisms were identified using single-stranded conformation polymorphism analysis. Possible functions of the protein based on homology to other members of the WD-repeat-containing family are discussed.

Linkage analyses of schizophrenia to chromosome 6p24-p22: an attempt to replicate.

The present study evaluates evidence for linkage of schizophrenia to chromosome 6p24-p22. An independent sample of 211 families ascertained on the basis of having an affected sib-pair diagnosed with schizophrenia or schizoaffective disorder was assessed with seventeen polymorphic markers spanning a 37cM region. Linkage analysis was performed with parametric and non-parametric methods to test for cosegregation using 4 models of inheritance. Neither two-point nor multipoint non-parametric analyses reached significance at a level less than 0.01 for any markers examined in the region and lod score analyses were not suggestive of linkage. Based on initial findings in the present data set and recently published linkage results, two specific areas were densely covered with markers and tested for linkage disequilibrium. After correcting for multiple comparisons within each locus, no significant deviation from expected allele transmission ratios was observed. The present findings together with the published literature fail to find consistent evidence of a linkage for schizophrenia to a single locus on chromosome 6.

Batched analysis of genotypes.

Polymorphic microsatellite markers are widely used in molecular analyses. The range of allele sizes and the allele frequencies within a population are important characteristics of the marker. Their determination previously has involved genotyping a large number of individuals. We have developed a technique for defining these characteristics by coamplification of many samples in a DNA pool. Groups of 32 and 42 DNA samples were genotyped and results were compared with those from individual genotype determinations. To improve the accuracy in the estimation of allele frequencies, arithmetic removal of stutter bands was carried out and the consistency of each marker was characterized. This approach was also applied to a group of 94 individuals. All of the work has been done using nonradioactive methods. Potential applications of this technique are in population genetics, high throughput genotyping, and loss of heterozygosity studies.

Vitamin D receptor alleles and rates of bone loss: influences of years since menopause and calcium intake.

A genetic marker for the 1,25-dihydroxyvitamin D receptor (VDR) is reported to account for much of the heritable component of bone density. It is not known whether VDR genotype influences bone accretion or loss, or how it is related to calcium metabolism. The VDR genotype was determined in 229 healthy postmenopausal women who previously participated in a calcium trial. VDR alleles were designated according to presence (b) or absence (B) of the BsmI restriction enzyme cutting site. There were 83 bb, 102 Bb, and 44 BB individuals. Two-thirds of the women took 500 mg of calcium supplement (mean calcium intake = 892 mg/day) and one-third a placebo (mean = 376 mg/day). Bone mineral density (BMD) at the femoral neck, spine, and radius were measured by dual- and single-photon absorptiometry at baseline and after 1 and 2 years. Among women more than 10 years postmenopausal, those with the BB genotype had the lowest femoral neck BMD. Rates of bone loss over 2 years were greater in the BB group at all sites (e.g., at the femoral neck, bb, 0.45 +/- 0.43; Bb, -0.01 +/- 0.40; BB, -0.99 +/- 0.50%/year; BB vs. bb, p = 0.01), and this trend was found both in women < 10 years since menopause (e.g., at the radius, bb, 0.43 +/- 0.47; Bb, -0.37 +/- 0.42; BB, -1.20 +/- 0.59% per year; BB vs. bb, p = 0.02) and those > or = 10 years (radius, bb, -0.71 +/- 0.41; Bb, 0.08 +/- 0.39; BB, -1.41 +/- 0.49% per year; BB vs. Bb, p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Variability of dopamine D4 receptor (DRD4) gene sequence within and among nonhuman primate species.

The dopamine D4 receptor is one of five receptors known to function in mammalian dopaminergic pathways. The DNA sequence of the human dopamine D4 receptor gene (DRD4) has previously been investigated in several populations and found to be highly polymorphic at both the DNA and amino acid levels, exhibiting at least 25 alleles. This variation results from differences in the number and DNA sequence of a 48-bp (16-amino acid) repeat unit in the coding region of DRD4. In the present study, DRD4 DNA sequence was examined in at least two individuals from each of five nonhuman primate species. All five species exhibit intraspecies variability, including both single nucleotide substitutions and variation in the number of 48-bp repeat units. No differences were found between the two alleles of one individual from a sixth nonhuman species. Within each species, all of the DRD4 alleles share species-specific features, indicating that while repeat-unit variation is nearly ubiquitous, ancestral variation has been lost and subsequently regenerated in each of the evolutionary lineages studied. Chimpanzees and gorillas share a unique 12-bp deletion in the coding region of DRD4, outside the repeat-unit segment of the gene. This suggest that the extant chimpanzee DRD4 is more closely related to the gorilla DRD4 than either is to the human DRD4.

Computer-assisted restriction mapping: an integrated approach to handling experimental uncertainty.

Building a map of restriction sites from double-digest gel data can be a complex and frustrating task, especially when many DNA fragments are detected or when the gel results are ambiguous. 'Double Digester' is an interactive, graphical computer program which helps researchers understand and resolve such data. It explicitly represents the experimental data, the associated uncertainties, the researcher's hypotheses and possible map interpretations. Alternative solutions are frequently possible, and the differences between them may help determine which additional experiments might resolve ambiguities. Initial use has confirmed the benefits of this approach, and has suggested ways in which it can be refined and extended. Double Digester meets the need for a practical tool to help build restriction maps, and also illustrates how a computer-based tool can confront experimental uncertainty in an integrated fashion.

Alleles at the dopamine D4 receptor locus do not contribute to the genetic susceptibility to schizophrenia in a large Swedish kindred.

The discovery of a functional polymorphism within the dopamine D4 receptor gene (DRD4) has not only strengthened the hypotheses implicating DRD4 in the etiology of neuropyschiatric disorders, but also provided a genetic marker for testing these hypotheses. The possibility of the dopamine D4 receptor as a candidate gene for schizophrenia was investigated in a large Swedish kindred segregating for schizophrenia. Linkage to schizophrenia was tested by linkage analyses of 6 polymorphic markers (at 4 loci) in chromosome 11p15.5 including the dopamine D4 receptor (DRD4) and the tyrosine hydroxylase (TH) loci. Schizophrenia was excluded from close linkage to the DRD4 locus using two of the polymorphisms located within the dopamine D4 receptor gene. The first DRD4 polymorphism consists of variation in the number of a 48 bp imperfect direct repeat in the third exon; the second consists of a variable number of repeated G nucleotides in the first intron. In addition, some of the individuals homozygous for four or seven copies of 48 bp repeat alleles were tested for previously reported sequence variation among repeats. No single haplotype of the DRD4 alleles or haplotype of other markers in chromosome 11p15.5 was found to be common to the schizophrenic individuals in this family. Therefore, we find no evidence for linkage of the D4 receptor, or this region of 11p15.5, with genetic susceptibility to schizophrenia in this kindred.