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.

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.

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.

In vitro transcription analysis of the Drosophila tropomyosin and other muscle genes.

Nuclear transcription extracts were prepared from embryos of Drosophila melanogaster to study the in vitro transcription of the tropomyosin genes. Several non-muscle gene promoters, including the non-muscle promoter of the Tropomyosin II gene, were shown to be efficiently transcribed in vitro. The Tropomyosin I gene and the muscle promoter of the Tropomyosin II gene, as well as two other contractile protein muscle genes, were not transcribed in vitro. The embryonic extract did, however, contain developmental-specific proteins that bound to the muscle enhancer regulatory region of the Tropomyosin I gene.

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.

Aplastic anemia following infectious mononucleosis: possible immune etiology.

A 17-year-old female developed severe aplastic anemia following serologically proven infectious mononucleosis. In vitro studies, using the granulocyte colony forming technique, suggested that the aplasia may have resulted from an immune mechanism. The patient's marrow grew no granulocyte colonies and caused inhibition of colony formation when mixed with normal marrows. The patient recovered fully after therapy with antithymocyte globulin and marrow cultures showed disappearance of the inhibitory effect. These observations suggest that the severe aplasia may have resulted from an aberrant immune response which followed infection by EB virus.

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)

Presymptomatic testing using DNA markers for individuals at risk for familial multiple endocrine neoplasia 2A.

The carrier status of 39 at-risk individuals in 6 multiple endocrine neoplasia 2A families was determined using a DNA based test. We were able to calculate a virtual diagnosis (probability greater than 95%) for 77% of the individuals and a probable diagnosis (probability greater than 90%) for 90% of the individuals. This study points out some of the problems of specific pedigree structures that can affect the risk calculation. This study further shows that no single test based on either biochemistry, pathology, or genetics can consistently and unambiguously produce a presymptomatic diagnosis. We also describe two specific examples where DNA testing has helped to resolve clinical uncertainties in at-risk individuals.

The labeling of rabbit neutrophils with [111In]oxine.

We report here the successful labeling of rabbit peripheral blood neutrophils with [111In]oxine. We found that standard techniques for preparation of rabbit neutrophils, while acceptable for maintenance of in vitro function, rendered the neutrophils ineffective for in vivo use after labeling with 111In. Specifically, rabbit neutrophils were sensitive to the use of hypotonic shock for red cell elimination, centrifugation into a button during preparation, and the presence of oxine during chemotaxis in vitro. Using a carefully modified method of neutrophil preparation and labeling, we found that 111In-labeled rabbit neutrophils retained normal in vitro function, including chemotaxis. In addition, using our method, 34% +/- 5% of labeled neutrophils were recoverable in peripheral blood 5 min after intravenous injection. The half-life of circulating radio-labeled neutrophils was 5.6 +/- 2 h. Continuous external imaging of radio-labeled neutrophils after intravenous injection showed initial lung uptake, followed by rapid clearance of radioactivity in the lungs (50% clearance in 10.5 +/- 3.3 min.) Hepatic radioactivity was maximal by 30 min after injection and thereafter slowly declined. Finally, we found that 111In-labeled rabbit neutrophils migrated to sites of artificially induced inflammation. Our findings indicate that 111In-labeled rabbit neutrophils, if prepared under optimal conditions, should provide a useful tool for investigating the fate of neutrophils in experimental inflammatory conditions in this animal.

Tension ball valve mucus plug in asthma.

This report describes a patient with status asthmaticus and respiratory failure in whom profound hypoxemia developed during mechanical ventilation. During the hypoxemic episode, breath sounds were absent over the left lung, and chest radiography revealed a hyperlucent left hemithorax with tension shift of the mediastinum to the right. The presence of lung markings in the left lung on radiography eliminated the possibility of tension pneumothorax and led to the diagnosis of tension mediastinal shift secondary to a ball valve obstruction by a central mucus plug. Bronchoscopic lung lavage removed the mucus plug, thereby correcting the hypoxemia. Recognition of this previously undescribed acute complication of mechanical ventilation in status asthmaticus is essential so that confusion with tension pneumothorax is avoided and appropriate therapy instituted.

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.

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.

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.

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.

Kinetic and morphological changes induced in human blood leucocytes by cytochalasin D and E.

The effect of increasing concentrations of cytochalasin D and E, up to toxicity, on the velocity of blood leucocytes from normal subjects was measured in vitro using a high-resolution objective and phase-contrast time-lapse photography. The dose-response effect for the two different cytochalasins differed in accordance with the different cell specificity of their membrane binding. The average velocity of granulocytes was reduced at cytochalasin D concentrations above 5 x 10(-7)M and cytochalasin E concentrations above 5 x 10(-5)M. The effect on monocytes and eosinophils was similar. In contrast the velocity of lymphocytes was not affected until cytotoxic concentrations were reached. The concentration ranges which inhibited locomotion corresponded well with the concentration ranges of the cytochalasins which have an in vitro effect on microfilaments. The concentrations which induced additional morphological changes in lymphocytes also correlate well with the concentrations found to inhibit cross-linking in vitro, as well as those known to induce morphological changes in, for example, fibroblasts in vivo. Cytotoxic effects were first observed with ten-fold higher concentrations of cytochalasin E than of cytochalasin D.

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.

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.