Detection of Tyrosinase Autoantibodies in Patients With Vitiligo Using 35S-Labeled Recombinant Human Tyrosinase in a Radioimmunoassay

Tyrosinase antibodies recently have been reported to occur frequently in patients with vitligo. We describe the detection of tyrosinase antibodies in vitiligo patients using in vitro 35S-labeled human tyrosinase in a radioimmunoassay. Of 46 vitiligo sera examined in the assay, five (10.9%) were found to be positive for tyrosinase antibodies. In contrast, 20 control sera and sera from 10 patients with Hashimoto's thyroiditis were negative. Four of the sera positive in the radioimmunoassay were also positive in an ELISA using mushroom tyrosinase as antigen. Absorption studies indicated that pre-incubation with mushroom tyrosinase absorbed out the immunoreactivity of the positive sera in the radioimmunoassay, suggesting cross-reactivity, but this absorption was never complete, indicating that there are tyrosinase antibodies in human sera that do not react with the mushroom protein. There was no obvious association between the presence of tyrosinase antibodies and the age of the patients (range: 22–62 y), their duration of disease (range: 5–20 y), or the type of vitiligo (one segmental, one symmetricallperiorificial, three symmetrical), although the three patients with the highest antibody levels also had an associated autoimmune disorder (one with Graves' disease; two with autoimmune hypothyroidism). The results confirm that tyrosinase autoantibodies are present in the sera of vitiligo patients but at a low frequency. The technique described is sensitive and quantitative and allows the detection of confirmational epitopes. It will be useful in longitudinal studies to determine the relation between the clinical features of vitiligo and tyrosinase antibody levels

Autoantigens in Vitiligo Identified by the Serological Selection of a Phage-Displayed Melanocyte cDNA Expression Library

Vitiligo is an acquired idiopathic hypomelanotic disorder characterized by circumscribed depigmented macules resulting from the loss of cutaneous melanocytes. Although the exact cause of vitiligo remains obscure, autoimmunity may play a role in the development of the disease. The present study was undertaken to investigate the applicability of phage display technology to identify B-cell autoantigens in vitiligo. A melanocyte cDNA phage display library was subjected to rounds of enrichment with vitiligo patient IgG. Subsequently, enriched IgG-binding peptides representing putative autoantigens were identified by sequencing their encoding cDNAs. Radioimmunoassays were used to confirm the immunoreactivity of vitiligo patient (n=61) and control (n=28) sera to several of the putative autoantigens. Non-segmental vitiligo patient sera (n=53) were positive for antibody (Ab) reactivity to gamma-enolase (8%); alpha-enolase (9%); heat-shock protein 90 (13%); osteopontin (4%); ubiquitin-conjugating enzyme (15%); translation-initiation factor 2 (6%); and GTP-binding protein, Rab38 (15%). Ab reactivity to at least one of the previously unknown autoantigens was detected in 51% of patients with non-segmental vitiligo. In contrast, Ab reactivity in a group of patients with segmental vitiligo (n=8) was not demonstrated. Overall, the study indicated that the targets of autoantibodies in vitiligo patients can be revealed by employing the methodology of phage display

Mapping of Human Autoantibody Binding Sites on the Calcium-Sensing Receptor

Previously, we have demonstrated the presence of anti-calcium-sensing receptor (CaSR) antibodies in patients with autoimmune polyglandular syndrome type 1 (APS1), a disease that is characterized in part by hypoparathyroidism involving hypocalcemia, hyperphosphatemia, and low serum levels of parathyroid hormone. The aim of this study was to define the binding domains on the CaSR of anti-CaSR antibodies found in APS1 patients and in one patient suspected of having autoimmune hypocalciuric hypercalcemia (AHH). A phage-display library of CaSR peptides was constructed and used in biopanning experiments with patient sera. Selectively enriched IgG-binding peptides were identified by DNA sequencing, and subsequently, immunoreactivity to these peptides was confirmed in ELISA. Anti-CaSR antibody binding sites were mapped to amino acid residues 41–69, 114–126, and 171–195 at the N-terminal of the extracellular domain of the receptor. The major autoepitope was localized in the 41–69 amino acid sequence of the CaSR with antibody reactivity demonstrated in 12 of 12 (100%) APS1 patients with anti-CaSR antibodies and in 1 AHH patient with anti-CaSR antibodies. Minor epitopes were located in the 114–126 and 171–195 amino acid domains, with antibody reactivity shown in 5 of 12 (42%) and 4 of 12 (33%) APS1 patients, respectively. The results indicate that epitopes for anti-CaSR antibodies in the AHH patient and in the APS1 patients who were studied are localized in the N-terminal of the extracellular domain of the receptor. The present work has demonstrated the successful use of phage-display technology in the discovery of CaSR-specific epitopes targeted by human anti-CaSR antibodies. © 2010 American Society for Bone and Mineral Research

Nuclear factors involved in mitochondrial translation cause a subgroup of combined respiratory chain deficiency.

Mutations in several mitochondrial DNA and nuclear genes involved in mitochondrial protein synthesis have recently been reported in combined respiratory chain deficiency, indicating a generalized defect in mitochondrial translation. However, the number of patients with pathogenic mutations is small, implying that nuclear defects of mitochondrial translation are either underdiagnosed or intrauterine lethal. No comprehensive studies have been reported on large cohorts of patients with combined respiratory chain deficiency addressing the role of nuclear genes affecting mitochondrial protein synthesis to date. We investigated a cohort of 52 patients with combined respiratory chain deficiency without causative mitochondrial DNA mutations, rearrangements or depletion, to determine whether a defect in mitochondrial translation defines the pathomechanism of their clinical disease. We followed a combined approach of sequencing known nuclear genes involved in mitochondrial protein synthesis (EFG1, EFTu, EFTs, MRPS16, TRMU), as well as performing in vitro functional studies in 22 patient cell lines. The majority of our patients were children (<15 years), with an early onset of symptoms <1 year of age (65%). The most frequent clinical presentation was mitochondrial encephalomyopathy (63%); however, a number of patients showed cardiomyopathy (33%), isolated myopathy (15%) or hepatopathy (13%). Genomic sequencing revealed compound heterozygous mutations in the mitochondrial transfer ribonucleic acid modifying factor (TRMU) in a single patient only, presenting with early onset, reversible liver disease. No pathogenic mutation was detected in any of the remaining 51 patients in the other genes analysed. In vivo labelling of mitochondrial polypeptides in 22 patient cell lines showed overall (three patients) or selective (four patients) defects of mitochondrial translation. Immunoblotting for mitochondrial proteins revealed decreased steady state levels of proteins in some patients, but normal or increased levels in others, indicating a possible compensatory mechanism. In summary, candidate gene sequencing in this group of patients has a very low detection rate (1/52), although in vivo labelling of mitochondrial translation in 22 patient cell lines indicate that a nuclear defect affecting mitochondrial protein synthesis is responsible for about one-third of combined respiratory chain deficiencies (7/22). In the remaining patients, the impaired respiratory chain activity is most likely the consequence of several different events downstream of mitochondrial translation. Clinical classification of patients with biochemical analysis, genetic testing and, more importantly, in vivo labelling and immunoblotting of mitochondrial proteins show incoherent results, but a systematic review of these data in more patients may reveal underlying mechanisms, and facilitate the identification of novel factors involved in combined respiratory chain deficiency

Organic Reference Materials for Hydrogen, Carbon, and Nitrogen Stable Isotope-Ratio Measurements: Caffeines, n-Alkanes, Fatty Acid Methyl Esters, Glycines, L-Valines, Polyethylenes, and Oils

An international project developed, quality-tested, and determined isotope−δ values of 19 new organic reference materials (RMs) for hydrogen, carbon, and nitrogen stable isotope-ratio measurements, in addition to analyzing pre-existing RMs NBS 22 (oil), IAEA-CH-7 (polyethylene foil), and IAEA-600 (caffeine). These new RMs enable users to normalize measurements of samples to isotope−δ scales. The RMs span a range of δ^2H_(VSMOW-SLAP) values from −210.8 to +397.0 mUr or ‰, for δ^(13)C_(VPDB-LSVEC) from −40.81 to +0.49 mUr and for δ^(15)N_(Air) from −5.21 to +61.53 mUr. Many of the new RMs are amenable to gas and liquid chromatography. The RMs include triads of isotopically contrasting caffeines, C_(16) n-alkanes, n-C_(20)-fatty acid methyl esters (FAMEs), glycines, and L-valines, together with polyethylene powder and string, one n-C_(17)-FAME, a vacuum oil (NBS 22a) to replace NBS 22 oil, and a ^2H-enriched vacuum oil. A total of 11 laboratories from 7 countries used multiple analytical approaches and instrumentation for 2-point isotopic normalization against international primary measurement standards. The use of reference waters in silver tubes allowed direct normalization of δ2H values of organic materials against isotopic reference waters following the principle of identical treatment. Bayesian statistical analysis yielded the mean values reported here. New RMs are numbered from USGS61 through USGS78, in addition to NBS 22a. Because of exchangeable hydrogen, amino acid RMs currently are recommended only for carbon- and nitrogen-isotope measurements. Some amino acids contain ^(13)C and carbon-bound organic ^2H-enrichments at different molecular sites to provide RMs for potential site-specific isotopic analysis in future studies

What makes health impact assessments successful? Factors contributing to effectiveness in Australia and New Zealand

Background: While many guidelines explain how to conduct Health Impact Assessments (HIAs), less is known about the factors that determine the extent to which HIAs affect health considerations in the decision making process. We investigated which factors are associated with increased or reduced effectiveness of HIAs in changing decisions and in the implementation of policies, programs or projects. This study builds on and tests the Harris and Harris-Roxas' conceptual framework for evaluating HIA effectiveness, which emphasises context, process and output as key domains. Methods: We reviewed 55 HIA reports in Australia and New Zealand from 2005 to 2009 and conducted surveys and interviews for 48 of these HIAs. Eleven detailed case studies were undertaken using document review and stakeholder interviews. Case study participants were selected through purposeful and snowball sampling. The data were analysed by thematic content analysis. Findings were synthesised and mapped against the conceptual framework. A stakeholder forum was utilised to test face validity and practical adequacy of the findings. Results: We found that some features of HIA are essential, such as the stepwise but flexible process, and evidence based approach. Non-essential features that can enhance the impact of HIAs include capacity and experience; 'right person right level'; involvement of decision-makers and communities; and relationships and partnerships. There are contextual factors outside of HIA such as fit with planning and decision making context, broader global context and unanticipated events, and shared values and goals that may influence a HIA. Crosscutting factors include proactive positioning, and time and timeliness. These all operate within complex open systems, involving multiple decision-makers, levels of decision-making, and points of influence. The Harris and Harris-Roxas framework was generally supported. Conclusion: We have confirmed previously identified factors influencing effectiveness of HIA and identified new factors such as proactive positioning. Our findings challenge some presumptions about 'right' timing for HIA and the rationality and linearity of decision-making processes. The influence of right timing on decision making needs to be seen within the context of other factors such as proactive positioning. This research can help HIA practitioners and researchers understand and identify what can be enhanced within the HIA process. Practitioners can adapt the flexible HIA process to accommodate the external contextual factors identified in this report

The Long-Baseline Neutrino Experiment: Exploring Fundamental Symmetries of the Universe

The preponderance of matter over antimatter in the early Universe, the dynamics of the supernova bursts that produced the heavy elements necessary for life and whether protons eventually decay --- these mysteries at the forefront of particle physics and astrophysics are key to understanding the early evolution of our Universe, its current state and its eventual fate. The Long-Baseline Neutrino Experiment (LBNE) represents an extensively developed plan for a world-class experiment dedicated to addressing these questions. LBNE is conceived around three central components: (1) a new, high-intensity neutrino source generated from a megawatt-class proton accelerator at Fermi National Accelerator Laboratory, (2) a near neutrino detector just downstream of the source, and (3) a massive liquid argon time-projection chamber deployed as a far detector deep underground at the Sanford Underground Research Facility. This facility, located at the site of the former Homestake Mine in Lead, South Dakota, is approximately 1,300 km from the neutrino source at Fermilab -- a distance (baseline) that delivers optimal sensitivity to neutrino charge-parity symmetry violation and mass ordering effects. This ambitious yet cost-effective design incorporates scalability and flexibility and can accommodate a variety of upgrades and contributions. With its exceptional combination of experimental configuration, technical capabilities, and potential for transformative discoveries, LBNE promises to be a vital facility for the field of particle physics worldwide, providing physicists from around the globe with opportunities to collaborate in a twenty to thirty year program of exciting science. In this document we provide a comprehensive overview of LBNE's scientific objectives, its place in the landscape of neutrino physics worldwide, the technologies it will incorporate and the capabilities it will possess.Comment: Major update of previous version. This is the reference document for LBNE science program and current status. Chapters 1, 3, and 9 provide a comprehensive overview of LBNE's scientific objectives, its place in the landscape of neutrino physics worldwide, the technologies it will incorporate and the capabilities it will possess. 288 pages, 116 figure

Sulforaphane restores cellular glutathione levels and reduces chronic periodontitis neutrophil hyperactivity in vitro

The production of high levels of reactive oxygen species by neutrophils is associated with the local and systemic destructive phenotype found in the chronic inflammatory disease periodontitis. In the present study, we investigated the ability of sulforaphane (SFN) to restore cellular glutathione levels and reduce the hyperactivity of circulating neutrophils associated with chronic periodontitis. Using differentiated HL60 cells as a neutrophil model, here we show that generation of extracellular O2 . - by the nicotinamide adenine dinucleotide (NADPH) oxidase complex is increased by intracellular glutathione depletion. This may be attributed to the upregulation of thiol regulated acid sphingomyelinase driven lipid raft formation. Intracellular glutathione was also lower in primary neutrophils from periodontitis patients and, consistent with our previous findings, patients neutrophils were hyper-reactive to stimuli. The activity of nuclear factor erythroid-2-related factor 2 (Nrf2), a master regulator of the antioxidant response, is impaired in circulating neutrophils from chronic periodontitis patients. Although patients' neutrophils exhibit a low reduced glutathione (GSH)/oxidised glutathione (GSSG) ratio and a higher total Nrf2 level, the DNA-binding activity of nuclear Nrf2 remained unchanged relative to healthy controls and had reduced expression of glutamate cysteine ligase catalytic (GCLC), and modifier (GCLM) subunit mRNAs, compared to periodontally healthy subjects neutrophils. Pre-treatment with SFN increased expression of GCLC and GCM, improved intracellular GSH/GSSG ratios and reduced agonist-activated extracellular O2 . - production in both dHL60 and primary neutrophils from patients with periodontitis and controls. These findings suggest that a deficiency in Nrf2-dependent pathways may underpin susceptibility to hyper-reactivity in circulating primary neutrophils during chronic periodontitis. © 2013 Dias et al