Performance of a polymer coated silicon microarray for simultaneous detection of food allergen-specific IgE and IgG4.

BACKGROUND: Microarray-based component-resolved diagnostics (CRD) has become an accepted tool to detect allergen-specific IgE sensitization towards hundreds of allergens in parallel from one drop of serum. Nevertheless, specificity and sensitivity as well as a simultaneous detection of allergen-specific IgG4 , as a potential parameter for tolerance development, remain to be optimized. OBJECTIVE: We applied the recently introduced silicon chip coated with a functional polymer named copoly(DMA-NAS-MAPS) to the simultaneous detection of food allergen-specific IgE and IgG4 , and compared it with ImmunoCAP and ImmunoCAP ISAC. Inter- and intraslide variation, linearity of signal and working range, sensitivity and application of internal calibrations for IgE and IgG4 were assessed. METHODS: Native and recombinant allergenic proteins from hen's egg and cow's milk were spotted on silicon chips coated with copoly(DMA-NAS-MAPS) along with known concentrations for human IgE and IgG4 . A serum pool and 105 patient samples were assessed quantitatively and semi-quantitatively with the ImmunoCAP and ImmunoCAP ISAC and correlated with IgE- and IgG4 -specific fluorescence on silicon microarrays. RESULTS: Allergen-specific IgE and IgG4 were detected in parallel using two fluorescent dyes with no crosstalk. Results from the ImmunoCAP correlated better with microarray fluorescence than with ImmunoCAP ISAC except for the allergen ovomucoid. The working range of the silicon microarray for total hen's egg-specific IgE was comparable to the range of 0.1 to >100 kUA /L of the ImmunoCAP system, whereas for total cow's milk, the silicon microarray was less sensitive. Detectable allergen-specific IgG4 could be determined only for low concentrations, but still correlated positively with ImmunoCAP results. CONCLUSIONS: We confirmed the ability of the polymer coated silicon microarray to be comparably sensitive to the ImmunoCAP ISAC for various food allergens. This suggests that the copoly(DMA-NAS-MAPS) microarray is a low-cost, self-producible alternative to the commercial ImmunoCAP ISAC in allergy research.

Natural tolerance development in cow's milk allergic children: IgE and IgG4 epitope binding.

BACKGROUND: Although most of cow's milk (CM) allergic children will outgrow their allergy, the pathomechanism of the natural development of tolerance remains poorly understood. It has been suggested that the balance between milk-specific IgE and IgG4 plays a major role. OBJECTIVE: We aimed to investigate differences in IgE and IgG4 antibody binding to CM epitopes between patients with persistent CM allergy (CMA) and those that naturally became tolerant. METHODS: Sera from 35 children with proven CMA (median age at inclusion of 10 months) were analyzed retrospectively; 22 patients have become tolerant (median age at tolerance acquisition of 51 months) during the study period as confirmed by a negative oral food challenge. IgE and IgG4 binding to sequential epitopes derived from five major CM proteins were measured with a peptide microarray-based immunoassay. RESULTS: At baselines, greater intensity and broader diversity of IgE and IgG4 binding have been found in children with persistent CMA beyond 5 years of age compared to patients with transient CMA. Moreover, children with transient CMA had IgE and IgG4 antibodies that more often recognized the same epitopes, compared to those with persistent CMA. From baseline to the time of tolerance development, both IgE and IgG4 binding intensity decreased significantly, particularly in areas of α-s- and ß-casein (P<.01, false discovery rate [FDR]<.1). Interestingly, differences between IgE and IgG4 binding intensity to CM peptides decreased when the patients became tolerant. CONCLUSIONS: Our results suggest that the overlap between IgE and IgG4 might be important in natural tolerance acquisition. Further studies are needed to confirm our data and can eventually lead to development of more targeted treatment of food allergy.

A prolonged outbreak of Salmonella Infantis associated with pork products in central Germany, April-October 2013.

One of the largest and longest Salmonella outbreaks in Germany within the last 10 years occurred in central Germany in 2013. To identify vehicles of infection, we analysed surveillance data, conducted a case-control study and food traceback. We identified 267 cases infected with Salmonella Infantis with symptom onset between 16 April and 26 October 2013 in four neighbouring federal states. Results of our study indicated that cases were more likely to have eaten raw minced pork from local butcher's shops [odds ratio (OR) 2·5, 95% confidence interval (CI) 1·1-5·8] and have taken gastric acid-reducing or -neutralizing medication (OR 3·8, 95% CI 1·3-13) than controls. The outbreak was traced back to contaminated raw pork products found in different butcher's shops supplied by one slaughterhouse, to pigs at one farm and to an animal feed producer. Characterization of isolates of human, food, animal, feed, and environmental origin by phage-typing and pulsed-field gel electrophoresis confirmed the chain of infection. Insufficient hygiene standards in the slaughterhouse were the most probable cause of the ongoing transmission. We recommend that persons taking gastric acid suppressants should refrain from consuming raw pork products. Improving and maintaining adequate hygiene standards and process controls during slaughter is important to prevent future outbreaks.

Positive reactions to placebo in children undergoing double-blind, placebo-controlled food challenge.

BACKGROUND: The gold standard in the diagnosis of food allergy is the double-blind, placebo-controlled oral food challenge (DBPCFC). During this challenge, patients receive the allergenic food and placebo on separate randomized days, while being monitored for clinical reactions. Interestingly, some reactions are assessed as positive although the patients had received placebo. The aim of our study was to analyze incidence and characteristics of positive placebo reactions during DBPCFCs. METHODS: In food-allergic children, we retrospectively analyzed positive placebo reactions in DBPCFCs in 740 placebo challenges in our department. Individual characteristics were compared, such as age or IgE levels, as well as clinical symptoms. RESULTS: Of all placebo challenges, 2.8% (21 of 740) were assessed as positive. Young children (age ≤ 1.5 years) had more (P = 0.047) positive placebo challenges (4.0%) compared to older children (age > 1.5 years; 1.5%). Children with positive placebo challenges had higher levels of total IgE (median 201 kU/L) compared to negatively classified children (median 110 kU/L). In children with positive placebo reactions, skin symptoms were observed significantly more often, with a worsening of atopic eczema (AE) as the most reported symptom. CONCLUSION: Placebo reactions in DBPCFC are not common. Worsening of AE is the most frequent clinical reaction associated with positive placebo challenges, and young children (age ≤ 1.5 years) seem to be affected more often. Therefore - contrary to current recommendations - DBPCFC tests should be considered in infants and young children, especially those with a history of AE.

Land use effects on phosphorus sequestration in soil aggregates in western Iran.

Cultivating native lands may alter soil phosphorus (P) distribution and availability. The present study aimed to determine the distribution of P in soil aggregates for different long-term land management practices. The partitioned P in labile (L), Fe/Al-bound, Ca-bound, organic pools, and total P in four aggregate size fractions were determined for five land uses (forest, vineyard after 30 years, wetland, alfalfa, and wheat cultivated soil after 20 years). Both native land uses (forest and wetland) were distinguished by high and low amounts of large macro- and micro-aggregates, respectively, compared with disturbed soils (vineyard, alfalfa, and wheat soils). Labile P in large macro-aggregates were higher in native land use when compared with the other land uses, which led to increasing lability of P and accelerated water pollution. Soils under native conditions sequestered more Ca-bound P in large macro-aggregates than the soils in disturbed conditions. Conversion of native lands to agricultural land caused enhanced organic P storage in aggregates smaller than the 2 mm from 31.0 to 54.3%. Soils under forest had 30% total P more than the vineyard for the aggregates >2 mm after 30 years land use change. However, the amount of P in smaller (<2 mm) sized aggregates was increased by 29% for the vineyard when compared with the forest. The P storage as bound Ca particles for the large macro-aggregates had negative correlation with the micro-aggregates.

Peanut allergen in house dust of eating area and bed--a risk factor for peanut sensitization?

It has been hypothesized that high environmental exposure to peanut allergens may be a potent risk factor for cutaneous sensitization. Therefore, we wanted to investigate whether peanut proteins are detectable in house dust of different household areas. Peanut levels of dust samples were measured with ELISA. Overall, peanut was detectable in 19 of 21 households in the eating area and/or in bed. The frequency of peanut consumption correlated with peanut levels. Forty-eight hours after intentional peanut consumption, peanut levels were highly increased. Nevertheless, further research is required to prove whether peanut allergen in house dust can cause sensitization via skin.

Chemometrics in Forensic Chemistry - Part III: Quality assessment and interpretation of chemometric output.

The number of samples sent to forensic laboratories as well as the complexity of the drug situation has increased tremendously during recent years. At the same time the amount of data gathered from chemical measurements has been mounting. This creates challenges for forensic chemists: how to handle the data, how to reliably answer the questions asked, and how to examine the data to find new properties or how to disclose connections with respect to source attribution of samples within a case or retrospective to past cases, stored in a database. Previously published articles Chemometrics in Forensic Chemistry - Part I and II discussed where in the forensic workflow of routine casework chemometrics is applied, and presented examples of chemometric methods used in cases of illicit drugs. This article explains through examples that the chemometric results must never stand-alone. Before such results are reported, quality assessment steps, which may consist of operational, chemical, and forensic assessments are required. In each case a forensic chemist needs to consider the suitability of chemometric methods, based on their strengths, weaknesses, opportunities and threats (SWOT). This is because while chemometric methods are powerful tools managing complex data, they are to some extent chemically blind.

Individual cow's milk allergens as prognostic markers for tolerance development?

BACKGROUND: Cow's milk allergy (CMA) is one of the most common causes of food allergy in the first years of life. Fortunately, the majority of children with CMA develop clinical tolerance with time. However, no good individual markers exist to predict whether this will occur. Therefore, a prognosis to identify children with persistent CMA at diagnosis would be helpful. OBJECTIVE: In this study, we sought to assess whether measurement of IgE to individual allergens of cow's milk (CM) would separate patients with persistent CMA from those who became clinically tolerant to CM over time. METHODS: A total of 52 patients ranging from 3 months to 114 months of age with proven CMA by DBPCFC were followed over time. From these 52 patients, 32 (61.5%) patients became tolerant in the analysed time period. All patients were rechallenged at least once, some were rechallenged two or three times. Serum was analysed prior to each challenge for specific IgE, IgG and IgG4 binding to crude CM protein as well as to individual allergens of CM. RESULTS: The individual likelihood of outgrowing CMA significantly correlates with a low level of CM-specific IgE as well as a low level of specific IgE to α-lactalbumin, ß-lactoglobulin (Bos d5.0102), κ-casein and α(s1) -casein. No significant correlation was found for IgE levels to total casein, lactoferrin, ß-casein and ß-lactoglobulin (Bos d5.0101) as well as IgG and IgG4 levels to α-lactalbumin, ß-lactoglobulin and total casein. CONCLUSIONS: CM-specific IgE is a good prognostic marker for persistence of CMA. In addition, component-resolved diagnostic showed similar results. However, in our view, the rising laboratory costs do not justify a measurement on a daily basis. Additional determination of specific IgG or IgG4 levels was not useful in predicting tolerance development in our study population.

Crystallization behavior of rare-earth doped fluorochlorozirconate glasses.

A series of fluorochlorozirconate (FCZ) glasses, each doped with a different rare-earth, was prepared and examined to determine thermal stability and activation energy, Ea , of the dopant dependent BaCl2 crystallization. Non-isothermal differential scanning calorimetry (DSC) measurements were done to investigate the endothermic and exothermic reactions upon heat treatment of the glass samples. In comparison to the rare-earth free FCZ glass, significant changes in the Hruby constant, Hr , and Ea were found due to the addition of a rare-earth and also between the individual dopants.

Glutathione peroxidase-2 protects from allergen-induced airway inflammation in mice.

The aim of the present study was to identify and validate the biological significance of new genes/proteins involved in the development of allergic airway disease in a murine asthma model. Gene microarrays were used to identify genes with at least a two-fold increase in gene expression in lungs of two separate mouse strains with high and low allergic susceptibility. Validation of mRNA data was obtained by western blotting and immunohistochemistry, followed by functional analysis of one of the identified genes in mice with targeted disruption of specific gene expression. Expression of two antioxidant enzymes, glutathione peroxidase-2 (GPX2) and glutathione S-transferase omega (GSTO) 1-1 was increased in both mouse strains after induction of allergic airway disease and localised in lung epithelial cells. Mice with targeted disruption of the Gpx-2 gene showed significantly enhanced airway inflammation compared to sensitised and challenged wild-type mice. Our data indicate that genes encoding the antioxidants GPX2 and GSTO 1-1 are common inflammatory genes expressed upon induction of allergic airway inflammation, and independently of allergic susceptibility. Furthermore, we provide evidence to illustrate the importance of a single antioxidant enzyme, GPX2, in protection from allergen-induced disease.

Lipocalin2 protects against airway inflammation and hyperresponsiveness in a murine model of allergic airway disease.

BACKGROUND: Allergen-induced bronchial asthma is a chronic airway disease that involves the interplay of various genes with environmental factors triggering different inflammatory pathways. OBJECTIVE: The aim of this study was to identify possible mediators of airway inflammation (AI) in a model of allergic AI via microarray comparisons and to analyse one of these mediators, Lipocalin2 (Lcn2), for its role in a murine model of allergic airway disease. METHODS: Gene microarrays were used to identify genes with at least a twofold increase in gene expression in the lungs of two separate mouse strains with high and low allergic susceptibility, respectively. Validation of mRNA data was obtained by Western blotting, followed by functional analysis of one of the identified genes, Lcn2, in mice with targeted disruption of specific gene expression. Epithelial cell cultures were undertaken to define induction requirements and possible mechanistic basis of the results observed in the Lcn2 knock-out mice. RESULTS: Lcn2 was up-regulated upon allergen sensitization and airway challenges in lung tissues of both mouse strains and retraced on the protein level in bronchoalveolar lavage fluids. Functional relevance was assessed in mice genetically deficient for Lcn2, which showed enhanced airway resistance and increased AI associated with decreased apoptosis of lung inflammatory cells, compared with wild-type controls. Similarly, application of Lcn2-blocking antibodies before airway challenges resulted in increased inflammation and reduced apoptosis. CONCLUSION: These data indicate a protective role for Lcn2 in allergic airway disease, suggesting a pro-apoptotic effect as the underlying mechanism.

The role of hen's egg-specific IgE, IgG and IgG4 in the diagnostic procedure of hen's egg allergy.

BACKGROUND: Hen's egg (HE) allergy is a common disease in childhood. HE-specific serum IgE has been correlated with the outcome of oral food challenge tests, and diagnostic decision points have been described as helpful but still not sufficient to reduce the requirement for oral food challenges. The aim of the study was to correlate HE-specific IgE, IgG and IgG4 levels with the outcome of double-blind, placebo-controlled food challenges (DBPCFC) in patients with suspected HE allergy to improve diagnostic procedures. METHODS: HE-specific IgE, IgG, and IgG4 levels were compared between 150 children with suspected HE allergy based on sensitization and/or patient's history who underwent DBPCFC. Sixty-six patients were HE-allergic (HE-sensitized with a positive DBPCFC), 48 HE-sensitized but tolerant (negative DBPCFC), and 36 patients were nonsensitized and tolerant (negative DBPCFC). Prior to food challenge HE-specific serum IgE, IgG, and IgG4 were measured with the Phadia CAP-system. RESULTS: HE-specific IgE was significantly higher in HE-allergic patients than in clinically tolerant ones. However, there was no difference in HE-specific IgG and IgG4 concentrations between the patient groups. CONCLUSION: A proposed cut-off level of 12 kU/l IgE would identify children above this level correctly as HE-allergic. The level of HE-specific IgG or IgG4 in serum of children with suspected HE allergy does not add any additional information in the diagnostic procedure of HE allergy. For diagnostic purposes, specific IgG or IgG4 should not routinely be tested.

Differential scanning calorimetry investigations on Eu-doped fluorozirconate-based glass ceramics.

The properties of Eu-doped fluorochlorozirconate (FCZ) glass ceramics upon thermal processing and the influence of Eu-doping on the formation of BaCl(2) nanocrystals therein have been investigated. Differential scanning calorimetry indicates that higher Eu-doping shifts the crystallization peak of the nanocrystals in the glass to lower temperatures, while the glass transition temperature remains constant. The activation energy and the thermal stability parameters for the BaCl(2) crystallization are determined.

Multi-functionality of fluorescent nanocrystals in glass ceramics.

Thermal processing of as-made fluorozirconate glasses, which were additionally doped with rare-earth and chlorine ions, results in the formation of fluorescent nanocrystals therein. For medical applications, the glasses were doped with divalent europium ions as the fluorescent rare-earth ion, while trivalent neodymium was used to develop up-conversion systems. The samples were annealed up to 290 °C to initiate the growth of hexagonal or orthorhombic phase BaCl2 nanocrystals therein. Upon annealing some of the rare-earth ions were incorporated into the BaCl2 nanocrystals leading to enhanced fluorescence properties. The particle diameters were in the range of a few nanometers to several tens of nanometers.

Chemometrics in forensic chemistry - Part II: Standardized applications - Three examples involving illicit drugs.

In the recently published article "Chemometrics in forensic chemistry - Part I: Implications to the forensic workflow" the application of chemometric methods in forensic casework was described. The steps to facilitate standardized chemometric procedures and the availability of chemometric tools such as software and a guideline are under development. Three examples of typical illicit drugs casework, wherein chemometric methods were applied, are presented in the current paper. The kind of questions presented in these examples cover identification, classification, comparison and quantification of illicit drugs. The examples include several types of data (low- or high-dimensional), pre-processing and chemometric analyses that are applied to answer the questions presented. The performance measures for the chemometric methods are described based on separate datasets for training and testing (validation) purposes. In this way it is illustrated how a chemometric method is set up and data analysis may be performed. The presented methods are intended to be easily translatable to questions in forensic chemistry that are not drug-related.

BCG priming of dendritic cells enhances T regulatory and Th1 function and suppresses allergen-induced Th2 function in vitro and in vivo.

BACKGROUND: The inverse correlation of mycobacterial infection with asthma prevalence and the inhibitory effects of vaccination with Bacille Calmette-Guérin (BCG) on airway hyperreactivity in asthma models suggest modulation of dendritic cell (DC) and T cell functions by mycobacterial compounds. METHODS: To delineate these immunological effects, the immunogenicity of BCG Copenhagen, BCG Chicago and BCG Pasteur was compared in a mouse model. Bone marrow-derived dendritic cells (BMDCs) from BALB/c mice were stimulated with ovalbumin (OVA) with or without BCG. BMDCs were phenotypically characterized by flow cytometry, and we used ELISA to measure the cytokine production of BMDCs as well as of co-cultivated allergen-specific T cells in response to OVA-pulsed. Immunomodulatory effects of BCG were studied in a model of allergic airway inflammation by adoptive transfer of allergen-pulsed BMDCs. RESULTS: Immunomodulation with BCG induced production of IL-10 and IL-12 by BMDCs. Co-cultured allergen-specific T cells produced less IL-5, IL-13 and IFN-gamma but more IL-10. Also the number of FoxP3(+) regulatory T cells was enhanced. Strongest effects were seen with BCG Chicago and BCG Pasteur. In vivo, administration of BCG modulated OVA-pulsed BMDCs then reduced eosinophilic airway inflammation but enhanced infiltration with granulocytes. Airway hyperreactivity and mucus production were reduced and more FoxP3(+) T cells were observed. CONCLUSION: BCG-induced suppression of Th2-type allergic airway inflammation was associated with enhancement of regulatory T cell function but also of Th1-associated neutrophilic airway inflammation. These findings raise concerns regarding the safety profile of BCG as a potential tool for prevention and therapy of allergic airway disease.

Upconverted fluorescence in Nd(3+)-doped barium chloride single crystals.

Upon excitation at 796 nm, Nd(3+)-doped BaCl(2) single crystals show several upconverted fluorescence bands in the visible spectral range, with the most intense bands at 530, 590 and 660 nm, in addition to the typical fluorescence bands in the infrared spectral range. The power dependence of the infrared fluorescence and the two-photon upconversion fluorescence intensities as well as the corresponding radiative lifetimes have been investigated. No 'saturation' of the fluorescence intensities was observed upon increasing the excitation power.

Chemometrics in forensic chemistry - Part I: Implications to the forensic workflow.

The forensic literature shows a clear trend towards increasing use of chemometrics (i.e. multivariate analysis and other statistical methods). This can be seen in different disciplines such as drug profiling, arson debris analysis, spectral imaging, glass analysis, age determination, and more. In particular, current chemometric applications cover low-dimensional (e.g. drug impurity profiles) and high-dimensional data (e.g. Infrared and Raman spectra) and are therefore useful in many forensic disciplines. There is a dominant and increasing need in forensic chemistry for reliable and structured processing and interpretation of analytical data. This is especially true when classification (grouping) or profiling (batch comparison) is of interest. Chemometrics can provide additional information in complex crime cases and enhance productivity by improving the processes of data handling and interpretation in various applications. However, the use of chemometrics in everyday work tasks is often considered demanding by forensic scientists and, consequently, they are only reluctantly used. This article and following planned contributions are dedicated to those forensic chemists, interested in applying chemometrics but for any reasons are limited in the proper application of statistical tools - usually made for professionals - or the direct support of statisticians. Without claiming to be comprehensive, the literature reviewed revealed a sufficient overview towards the preferably used data handling and chemometric methods used to answer the forensic question. With this basis, a software tool will be designed (part of the EU project STEFA-G02) and handed out to forensic chemist with all necessary elements of data handling and evaluation. Because practical casework is less and less accompanied from the beginning to the end out of the same hand, more and more interfaces are built in through specialization of individuals. This article presents key influencing elements in the forensic workflow related to the most meaningful chemometric application and evaluation.

Adjunctive lithium treatment in the prevention of suicidal behaviour in depressive disorders: a randomised, placebo-controlled, 1-year trial.

OBJECTIVE: Evidence based on controlled studies is still limited for treatment strategies that prevent recurrence of suicide attempts. Findings from observational as well as meta-analytic studies strongly suggest that lithium may have suicide-protective properties. METHOD: Patients with a recent suicide attempt in the context of an affective spectrum disorder (n = 167) were treated with either lithium or placebo during a 12-month period. RESULTS: Survival analysis showed no significant difference of suicidal acts between lithium and placebo-treated individuals (adjusted hazard ratio 0.517; 95% CI 0.18-1.43). However, post hoc analysis revealed that all completed suicides had occurred in the placebo group accounting for a significant difference in incidence rates (P = 0.049). CONCLUSION: Results indicate that lithium treatment might be effective in reducing the risk of completed suicide in adult patients with affective disorders. Our findings contribute to the growing body of evidence suggesting a specific antisuicidal effect of lithium.

Application of two-dimensional gas chromatography combined with pixel-based chemometric processing for the chemical profiling of illicit drug samples.

The chemical profiling of illicit drugs is a complex process. The results are affected by many different factors such as sample size, the sample processing conditions, the used analytical technique as well as the statistics that are applied. Within this proof-of-concept study, which was done in cooperation with the German Federal Criminal Police Office (Bundeskriminalamt, BKA), the adaptability of comprehensive two-dimensional (2D) gas chromatography (GCxGC) combined with a pixel-based chemometric data processing method is demonstrated. Samples of heroin and cannabis are extracted and analyzed with GCxGC-TOF-MS (time-of-flight mass spectrometry) and GCxGC-FID (flame ionization detection). The obtained second-order data are then used to identify possible marker compounds for the discrimination of the samples according to their chemical profile. The pixel-based chemometric process includes preprocessing steps (background correction, alignment of chromatograms and normalization) followed by an adaptation of hierarchical clustering to identify chemically similar samples, and finally a subsequent calculation of Fisher criterion based on the found clustering in order to identify promising marker compounds. The results of the pixel-based data analysis are compared to a limited peak-based study for cannabis and to a well-established standard method for the chemical profiling of heroin.