Long-Lasting Immune Responses 4 Years after GAD-Alum Treatment in Children with Type 1 Diabetes

A phase II clinical trial with glutamic acid decarboxylase (GAD) 65 formulated with aluminium hydroxide (GAD-alum) has shown efficacy in preserving residual insulin secretion in children and adolescents with recent-onset type 1 diabetes (T1D). We have performed a 4-year follow-up study of 59 of the original 70 patients to investigate long-term cellular and humoral immune responses after GAD-alum-treatment. Peripheral blood mononuclear cells (PBMC) were stimulated in vitro with GAD65. Frequencies of naïve, central and effector memory CD4+ and CD8+ T cells were measured, together with cytokine secretion, proliferation, gene expression and serum GAD65 autoantibody (GADA) levels. We here show that GAD-alum-treated patients display increased memory T-cell frequencies and prompt T-cell activation upon in vitro stimulation with GAD65, but not with control antigens, compared with placebo subjects. GAD65-induced T-cell activation was accompanied by secretion of T helper (Th) 1, Th2 and T regulatory cytokines and by induction of T-cell inhibitory pathways. Moreover, post-treatment serum GADA titres remained persistently increased in the GAD-alum arm, but did not inhibit GAD65 enzymatic activity. In conclusion, memory T- and B-cell responses persist 4 years after GAD-alum-treatment. In parallel to a GAD65-induced T-cell activation, our results show induction of T-cell inhibitory pathways important for regulating the GAD65 immunity

High spatial resolution analysis of ferromanganese concretions by LA-ICP-MS†

A procedure was developed for the determination of element distributions in cross-sections of ferromanganese concretions using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The effects of carrier flow rates, rf forward power, ablation energy, ablation spot size, repetition rate and number of shots per point on analyte intensity were studied. It is shown that different carrier gas flow rates are required in order to obtain maximum sensitivities for different groups of elements, thus complicating the optimisation of ICP parameters. On the contrary, LA parameters have very similar effects on almost all elements studied, thus providing a common optimum parameter set for the entire mass range. However, for selected LA parameters, the use of compromise conditions was necessary in order to compensate for relatively slow data acquisition by ICP-MS and maintain high spatial resolution without sacrificing the multielemental capabilities of the technique. Possible variations in ablation efficiency were corrected for mathematically using the sum of Fe and Mn intensities. Quantification by external calibration against matrix-matched standards was successfully used for more than 50 elements. These standards, in the form of pressed pellets (no binder), were prepared in-house using ferromanganese concentrates from a deep-sea nodule reference material as well as from shallow-marine concretions varying in size and having different proportions of three major phases: aluminosilicates, Fe- and Mn-oxyhydroxides. Element concentrations in each standard were determined by means of conventional solution nebulisation ICP-MS following acid digestion. Examples of selected inter-element correlations in distribution patterns along the cross-section of a concretion are given

Erratum: Corrigendum: Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution

International Chicken Genome Sequencing Consortium. The Original Article was published on 09 December 2004. Nature432, 695–716 (2004). In Table 5 of this Article, the last four values listed in the ‘Copy number’ column were incorrect. These should be: LTR elements, 30,000; DNA transposons, 20,000; simple repeats, 140,000; and satellites, 4,000. These errors do not affect any of the conclusions in our paper. Additional information. The online version of the original article can be found at 10.1038/nature0315

Multi-element analysis of environmental samples using LA-ICP-MS : method developments with emphasis on calibration strategies

Analytical applications based on the use of laser ablation-inductively coupled plasma-sector field mass spectrometry (LA-ICP-SFMS) have shown great potential for both quantitative and qualitative determination of elements in a variety of materials, although the weak point is finding a suitable calibration approach. The major focus of this work has, therefore, been internal standardisation and calibration. Methods for quantitative and qualitative analysis of geological (coal, ferromanganese concretions, sulphide minerals) and biological (nail and hair) samples have been developed. For coal, two reference materials were analysed after microwave (MW) treatment with different combinations of HNO3, aqua regia, HF and H2O2. Lithium metaborate (LMB) fusion with and without ashing of samples, as well as LA solid sampling on pressed coal pellets were also evaluated. External calibration for LA-ICP-SFMS was conducted using matrix-matched standard dopeds with standard solutions. This approach resulted in good accuracy for 46 out of 47 elements tested in the coal fly ash reference material. The precision was assessed in this work by replicate analyses of coal samples and was found to be, as average values for all elements, 4-5% and 10-15% relative standard deviation (RSD) for procedures involving digestion and LA sampling, respectively. For ferromanganese concretions, improvement of existing sample preparation schemes was necessary in order to expand the element coverage. The optimised and validated (for about 60 elements) method has the potential to replace the multitude of sample preparation and instrumental analytical techniques previously used to determine specific groups of elements in ferromanganese nodules. By using this improved method, it was possible to successfully quantify more than 50 elements by LA-ICP-SFMS using external calibration against matrix-matched standards. The results also show how different LA parameters affect the isotopic response and RSD. For sulphide minerals, LA-ICP-SFMS analysis was performed directly on polished crystalline sections. In sphalerite, a number of major and trace elements were determined by a ‘single-standard’ method using Zn for internal standardisation, together with correction for FeS impurities in the mineral, allowing straightforward quantification without using external methods for the determination of the actual Zn content. Results obtained by this approach were in good agreement with those from using external calibration. In order to evaluate the possibility to use crystalline sections of sphalerite and four other sulphide minerals as in-house standards, an analytical method was developed for multi-elemental analysis of such samples after preparation based on both MW digestion using different acid mixtures and LMB fusion. The performance of the sample preparation and analysis stages was evaluated using powdered pyrite and galena reference materials. It was found possible to use the selected minerals as matrix-matched standards for the determination of about 20 trace and ultra-trace elements by LA-ICP-SFMS. The second group of matrices concerned in this work comprises hair and nail samples. The first part of this study was focused on method validation for the determination of 71 elements by conventional ICP-SFMS analysis after MW-assisted digestion, with special attention paid to the correction of spectral interferences and to accuracy assessment. In the follow up study, the main concern was to evaluate the capabilities of LA-ICP-SFMS for quantitative multi-elemental analysis of hair and nail samples, emphasising the collection of spatially resolved information. Quantification was performed by means of an in-house, multi-element, matrix-matched standard using 32S+ as internal standard. A major conclusion of this thesis is that each sample type is in many respects unique, and therefore demands careful selection of a suitable calibration protocol, in order to ensure that reliable analytical results are delivered. The developed methods will make it possible to quantitatively analyse samples such as geological thin-sections, single sulphide grains, ferromanganese concretions, fingernails and hair. The results also show that LA-ICP-SFMS potentially can be used for obtaining quantitative analyses with good spatial resolution in a variety of materials.Godkänd; 2002; 20061110 (haneit

Multielemental analysis of geological and biological samples using laser ablation ICP-SFMS

The analytical performance of laser ablation (LA) for multielemental determination of geological and biological samples has been investigated. In the present study, LA has been used for sample introduction in inductively coupled plasma sector field mass spectrometry (ICP-SFMS). Although LA can provide semi-quantitative results rapidly and easily, the calibration process still remains the "Achilles heel" of the technique. The major goal of this study has been to investigate the capabilities of LA-ICP-SFMS for qualitative and quantitative analysis of various solid materials. LA was used for analyses of coal were calibration was performed by using coal powder doped with analyte elements. This was done by adding solution standards (including analyte and internal standard elements) to the coal, drying and finally homogenising, followed by pressing tablets. LA results were compared with data obtained by conventional solution nebulisation (SN) after preparation of coal samples using microwave (MW) digestion or fusion. In spite of a relatively poor agreement for elements such as As, Se, Sn, Re, Te, and Tl, accuracy obtained with LA in the present study is otherwise generally superior to previously reported data for LA and slurry nebulisation. For about 50 elements, results obtained with LA fall within 20% of those obtained by SN. The study shows the potential of LA quantification, based on solution-doped powders. However, the necessity of sample grinding and homogenising results in loss of spatial distribution information and makes the approach more vulnerable to sample contamination. The possibilities of sulphide minerals analysis by ICP-SFMS have been investigated. Seven elements (Co, Fe, Cd, Ag, Mn, Cu and S) have been quantitatively determined in sphalerite samples from the Zinkgruvan mine, using Zn as internal standard (IS). A straightforward calibration procedure allows on-line correction for possible Fe impurities at percent levels. Consequently, the use of complimentary techniques for determination of actual Zn content in the samples is avoided. The LA-ICP-SFMS results were compared with data from conventional SN introduction of sample solutions following acid digestion. Good agreement was found between the methods. For homogeneously distributed elements the overall precision for LA was found to be better than 10% RSD. A method for total mineral dissolution of five sulphides (sphalerite, pyrite, galena, pyrrhotite and chalcopyrite) has been developed, followed by multielemental analysis by ICP-SFMS. By performing this mineral characterisation the intention was to determine whether the analysed bulk mineral samples could be used as in-house LA calibration standards. The use of LA was focused towards elucidating whether the observed deviations in results obtained by ICP-SFMS for the two reference material powders used were caused by sample inhomogeneity or by inefficient matrix dissolution. It was found that the reference materials showed lack of accuracy in recommended concentrations for many trace and ultra trace elements, as well as possible inhomogeneity when using 50 mg sample amounts. The sulphide minerals studied appears to be suitable as matrix matched calibration standards for the determination of about 20 trace and ultra trace elements by LA. Laser ablation was also used for analysing element to sulphur ratios in washed human nails. The element ratios were then used to display the effectiveness of the applied washing method applied prior to MW digestion and analysis by ICP-SFMS. Based on the LA analysis, it was found that, even after the applied nail washing procedure, many elements are enriched in the surface of the nail.Godkänd; 2000; 20070317 (ysko

Multielement analysis of coal by ICP techniques using solution nebulization and laser ablation

The combination of inductively coupled plasma atomic emission spectrometry and high resolution inductively coupled plasma mass spectrometry for the determination of 70 elements in coal were studied. Four microwave- assisted digestion procedures with different dissolution mixtures (nitric and hydrofluoric acids, aqua regia and hydrogen peroxide), lithium metaborate fusion with and without previous sample ashing as well as direct sampling by laser ablation (LA) have been tested. Examples of spectral interferences are given and different correction procedures are discussed. Detection limits in the low ng g-1 range were obtained for most of the elements investigated by using high-purity reagents and by taking special care to prevent sample contamination during preparation. The precision was assessed from replicate analysis (including sample preparation) of coal samples and was found to be, as average values far all elements, 4-5% RSD and 10-15% RSD for procedures including sample digestion and LA sampling, respectively. The accuracy of the overall analytical procedures was estimated by analysis of certified reference materials and of a coal sample obtained from the Interlab Trace round robin test. Among the dissolution mixtures tested, the combination of nitric and hydrofluoric acids with hydrogen peroxide provide the best agreement with certified, recommended, literature-compiled or consensus values, though fusion is necessary to obtain quantitative recoveries for Si, Cr, Hf, W, Zr, Y. In general, results obtained by LA fall within ± 20% of those obtained after digestion

Next Generation Plasma Proteomics Identifies High-Precision Biomarker Candidates for Ovarian Cancer.

BACKGROUND: Ovarian cancer is the eighth most common cancer among women and has a 5-year survival of only 30-50%. The survival is close to 90% for patients in stage I but only 20% for patients in stage IV. The presently available biomarkers have insufficient sensitivity and specificity for early detection and there is an urgent need to identify novel biomarkers. METHODS: We employed the Explore PEA technology for high-precision analysis of 1463 plasma proteins and conducted a discovery and replication study using two clinical cohorts of previously untreated patients with benign or malignant ovarian tumours (N = 111 and N = 37). RESULTS: The discovery analysis identified 32 proteins that had significantly higher levels in malignant cases as compared to benign diagnoses, and for 28 of these, the association was replicated in the second cohort. Multivariate modelling identified three highly accurate models based on 4 to 7 proteins each for separating benign tumours from early-stage and/or late-stage ovarian cancers, all with AUCs above 0.96 in the replication cohort. We also developed a model for separating the early-stage from the late-stage achieving an AUC of 0.81 in the replication cohort. These models were based on eleven proteins in total (ALPP, CXCL8, DPY30, IL6, IL12, KRT19, PAEP, TSPAN1, SIGLEC5, VTCN1, and WFDC2), notably without MUCIN-16. The majority of the associated proteins have been connected to ovarian cancer but not identified as potential biomarkers. CONCLUSIONS: The results show the ability of using high-precision proteomics for the identification of novel plasma protein biomarker candidates for the early detection of ovarian cancer