Validation of an ELISA method for quantification of the major Timothy grass pollen allergen Phl p 5a (BSP090).

Progress towards standardisation of allergen products has been made in recent years. Nevertheless, no standardised test method to quantify the allergen content of grass pollen allergen products is available at present. One aim of the BSP090 project was to validate a quantitative assay for a major Timothy grass (Phleum pratense) pollen allergen, Phl p 5. Qualification of a candidate ELISA system was performed with regard to range, robustness and cross-reactivity in preliminary studies. The assay specifically detected Phl p 5 with a quantification range from 3.9 ng/mL to 62.5 ng/mL. Suitability to quantify recombinant and natural Phl p 5 was further assessed in a collaborative study including 14 laboratories in Europe and the USA. Precision and accuracy of the assay was satisfactory with 93% of calculated Phl p 5 concentrations and 100% of total recoveries being within the ± 30% acceptance range. Similar results were obtained for spike recoveries, with exclusion of the lowest concentration spike, showing spike recoveries exceeding the acceptance range for six laboratories. Inter-assay (repeatability) and inter-laboratory (reproducibility) variability were satisfactory, in the format used in the present study. Robustness towards different statistical methods for data analysis was demonstrated. In conclusion, the assay can easily be established in routine testing and results of the preliminary testing and collaborative study support the proposal of the assessed Phl p 5-specific ELISA as a European Pharmacopoeia general method.

Decision-making regarding oral nutritional supplements for nursing home residents with advanced dementia: A cross-sectional pilot study.

BACKGROUND: Surrogate decision-making regarding oral nutritional supplements (ONS) for nursing home residents with advanced dementia is a complex process. In this cross-sectional study, we assessed whether Dutch dietitians, elderly care physicians (physicians) and surrogate decision-makers (SDMs) differ in the factors that they regard important when considering ONS. We also investigated differences in opinion regarding whether or not ONS is a life-prolonging measure. METHODS: Through an online survey, 90 dietitians, 53 physicians and 70 SDMs of nursing home residents (all aged ≥ 65 years old with advanced dementia) rated the level of perceived influence of 11 pre-defined factors on their decision-making, ranked factors in order of importance and stated whether they considered ONS a life-prolonging measure or not. By statistical analysis, we tested differences in the mean sum of ranks for perceived influence differing between groups. We also tested differences in proportions between groups of those who considered ONS a life-prolonging measure. RESULTS: Rating of perceived influence significantly differed for six factors. Quality of life was ranked as the most influential factor by all groups. Dietitians significantly differed in their opinion on the life-prolonging effect of ONS from physicians (odds ratio = 0.29, 95% confidence interval = 0.13-0.65), as well as from SDMs (odds ratio = 0.22, 95% confidence interval = 0.10-0.45). CONCLUSIONS: Although all groups proclaimed quality of life to be first priority in decision-making, we found that Dutch dietitians, physicians and SDMs differed in what they regarded important when considering ONS for nursing home residents with advanced dementia. Regarding the life-prolonging effect of ONS, dietitians differed in opinion from physicians, as well as from SDMs.

The new latex allergen hev b 15: IgE-binding properties of a recombinant serine protease inhibitor

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Mycophenolate mofetil but not atorvastatin attenuates atherosclerosis in lupus-prone LDLr(-/-) mice.

RATIONALE: Recent clinical and preclinical studies have demonstrated that systemic lupus erythematosus (SLE) is associated with an increased risk for cardiovascular disease (CVD). However, unlike in the general population, little is known regarding the efficacy of atheroprotective interventions in patients with SLE. The current study aims to determine the benefit of lymphocyte inhibition on reducing the atherosclerotic burden in SLE-susceptible LDLr-deficient mice. METHODS: Female LDLr(-/-) mice were lethally irradiated and reconstituted with bone marrow from C57Bl/6 mice (LDLr.B6) or the SLE-susceptible B6.Sle1.2.3 mice (LDLr.Sle). At 16 weeks post transplant, mice were treated with atorvastatin (10 mg/kg), mycophenolate mofetil (MMF; 40 mg/kg), or both (MMF-A) for 8 weeks, after which the extent of atherosclerosis and the presence of SLE were assessed. RESULTS: Following 8 weeks of treatment, we observed that atorvastatin-mediated reduction in cholesterol levels attenuated atherogenesis in LDLr.B6 mice but failed to significantly reduce atherosclerotic lesion size in LDLr.Sle mice, in spite of a significant reduction in serum cholesterol levels. Treatment with MMF and MMF-A attenuated atherogenesis in LDLr.B6 and LDLr.Sle mice. In addition, MMF-containing regimens inhibited recruitment of CD4+ T cells to atherosclerotic lesions in LDLr.Sle mice. In these mice, MMF also reduced the proportion of activated splenic T cells, as well as interleukin 10 secretion by T cells. With regard to lupus activity, MMF had no overt effect on anti-double-stranded DNA (dsDNA) antibody titres or kidney function and pathology. CONCLUSIONS: The current study demonstrates that reduction of cholesterol levels alone is not atheroprotective in lupus-mediated atherogenesis. This is the first study to demonstrate that MMF reduces the atherosclerotic burden in a model of lupus-accelerated atherosclerosis. Our results suggest that MMF treatment may prove beneficial in preventing CVD in patients with SLE.

Microvascular and mitochondrial PO(2) simultaneously measured by oxygen-dependent delayed luminescence.

Measurement of tissue oxygenation is a complex task and various techniques have led to a wide range of tissue PO(2) values and contradictory results. Tissue is compartmentalized in microcirculation, interstitium and intracellular space and current techniques are biased towards a certain compartment. Simultaneous oxygen measurements in various compartments might be of great benefit for our understanding of determinants of tissue oxygenation. Here we report simultaneous measurement of microvascular PO(2) (µPO(2) ) and mitochondrial PO(2) (mitoPO(2) ) in rats. The µPO(2) measurements are based on oxygen-dependent quenching of phosphorescence of the near-infrared phosphor Oxyphor G2. The mitoPO(2) measurements are based on oxygen-dependent quenching of delayed fluorescence of protoporphyrin IX (PpIX). Favorable spectral properties of these porphyrins allow simultaneous measurement of the delayed luminescence lifetimes. A dedicated fiber-based time-domain setup consisting of a tunable pulsed laser, 2 red-sensitive gated photomultiplier tubes and a simultaneous sampling data-acquisition system is described in detail. The absence of cross talk between the channels is shown and the feasibility of simultaneous µPO(2) and mitoPO(2) measurements is demonstrated in rat liver in vivo. It is anticipated that this novel approach will greatly contribute to our understanding of tissue oxygenation in physiological and pathological circumstances.

Multiple wheat flour allergens and cross-reactive carbohydrate determinants bind IgE in baker's asthma.

BACKGROUND: Several wheat flour allergens relevant to baker's asthma have been identified in the last 25 years. The aim of this study was to determine the frequency of sensitization to these allergens in German bakers. METHODS: Using recombinant DNA technology, the following wheat flour allergens were cloned, expressed in Escherichia coli and purified: five subunits of the wheat α-amylase inhibitors (WTAI-CM1, WTAI-CM2, WTAI-CM3, WDAI-0.19 and WMAI-0.28), thioredoxin, thiol reductase or 1-cys-peroxiredoxin homologues, triosephosphate-isomerase, αß-gliadin, serpin, glyceraldehyde-3-phosphate-dehydrogenase, a nonspecific lipid transfer protein (nsLTP), dehydrin, profilin and peroxidase. In addition, ImmunoCAPs with the recombinant allergen ω-5-gliadin and two cross-reactive carbohydrate determinants (CCDs), horse radish peroxidase (HRP) and the N-glycan of bromelain (MUXF), were used. Specific IgE was measured in wheat flour-positive sera from 40 German bakers with work-related asthma/rhinitis and 10 controls with pollinosis. RESULTS: Thirty bakers (75%) had IgE to at least one of the 19 single allergens. Most frequent was IgE to WDAI-0.19, HRP and MUXF (25% each), followed by WTAI-CM1 (20%), thiol reductase (16%), WTAI-CM3 (15%), WTAI-CM2 and thioredoxin (12.5%), WMAI-28, triosephosphate-isomerase, αß-gliadin (10%), 1-cys-peroxiredoxin (7.5%), dehydrin, serpin, glyceraldehyde-3-phosphate-dehydrogenase (5%), ω-5-gliadin, nsLTP and profilin (2.5%). Fifteen bakers (38%) had IgE to any α-amylase inhibitor and 12 (30%) to at least one CCD. The controls reacted exclusively to CCDs (80%), profilin (60%), thioredoxin (30%), triosephosphate isomerase and nsLTP (10%). CONCLUSIONS: The single allergen sensitization profiles obtained with 17 recombinant wheat flour allergens and two CCDs revealed no major allergen for German bakers. The highest frequencies were found for α-amylase inhibitors and CCDs.

Allergen content of grass pollen preparations for skin prick testing and sublingual immunotherapy.

BACKGROUND: The allergen content of diagnostics and immunotherapeutics is crucial for effective diagnosis and treatment. The aim of this study was to quantify and compare the allergen content of different grass pollen preparations for skin prick testing and sublingual immunotherapy (SLIT). METHODS: Five skin prick test (SPT) solutions and 10 sublingual immunotherapeutics were analysed for protein and allergen concentration by Bradford assay, inhibition of IgE-binding to Phleum pratense ImmunoCAPs and content of the main allergen Phl p 5 by two-site enzyme immunoassay. In addition, the grass pollen preparations were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analyses. RESULTS: Protein concentrations of SPT solutions ranged from 15 to 427 microg/ml, and Phl p 5 concentrations ranged from 0.15 to 18.3 microg/ml. The ranking of SPT solutions concerning Phl p 5 content and IgE inhibition capacity was the same, and the ranking of protein and allergen content was closely correlated (r = 0.9). Protein content of the maintenance doses of the immunotheurapeutics ranged from 5 to 153 microg, Phl p 5 content ranged from 0.2 to 21.6 microg. IgE inhibition capacity of the maintenance doses was closely correlated to their Phl p 5 and protein content. SDS-PAGE and immunoblots confirmed the differences in protein and allergen content. CONCLUSIONS: Grass pollen preparations for SPT and SLIT varied greatly concerning protein and allergen content. Whereas this result corresponds to previous analyses results of SPT solutions, it was the first comparison of grass pollen immunotherapeutics. For diagnosis and therapy, these differences should be taken into account.

Occupational hypersensitivity pneumonitis due to molds in an onion and potato sorter.

A 42-year-old female non-smoking onion and potato sorter developed work-related shortness of breath, cough, fatigue and flu-like symptoms. The diagnosis of hypersensitivity pneumonitis was based on patchy infiltrates in both lungs on high resolution computed tomography and lymphocytosis of 71% in a bronchoalveolar lavage sample with a CD4/CD8 ratio of 0.4. Exposure cessation and initial corticosteroid therapy resulted in complete recovery. IgG antibodies to Penicillium species and Fusarium solani cultivated from samples from the patient's workplace were detected in the patient's serum and cross-reactivity was demonstrated within Penicillium species, but also between Penicillium species and Aspergillus fumigatus. We conclude that occupational hypersensitivity pneumonitis due to molds may develop in onion and potato sorters.

Identification of an obeche (Triplochiton scleroxylon) wood allergen as a class I chitinase.

BACKGROUND: Wood dust is known to cause allergic occupational asthma and obeche (Triplochiton scleroxylon) is a prominent exponent in this field. However, the knowledge about wood allergens is still limited. The aim of this study was to identify and characterize obeche wood allergens. METHODS: Obeche extracts were prepared from freshly ground in comparison to 7 years stored wood dust and investigated by Sodium dodecyl sulfate-polyacrylamid gel electrophoresis, enzyme-linked allergosorbent test and immunoglobulin (Ig)E-immunoblot. Allergens were detected by specific IgE of seven obeche allergic patients' sera and protein analysis was performed by mass spectrometry. Cross-reactivity was demonstrated by ImmunoCAP-inhibition with sera of seven obeche and four latex-allergic patients. RESULTS: Obeche extracts showed different protein pattern and IgE-binding capacities depend on the age of the wood dust. A 38 kDa protein was identified as major obeche wood allergen, detected by six of seven (85%) obeche allergic patients' sera and was entitled as Trip s 1. Trip s 1 is homologous to plant class I chitinases and exhibited enzyme activity demonstrated by chitinolysis. Co-recognition or cross-reactivity of Trip s 1 according to structural similarity was seen in sera of latex allergic patients. IgE inhibition studies with obeche as solid phase and Trip s 1 and latex hevein as inhibitor demonstrated that Trip s 1 was a more effective inhibitor in obeche as well as in latex allergic patients' sera. CONCLUSIONS: Trip s 1 is a new obeche wood allergen of the plant class I chitinase family. This finding may explain the dominant role of obeche in sensitization against wood dust.

Differentiation between cosensitization and cross-reactivity in wheat flour and grass pollen-sensitized subjects.

BACKGROUND: To diagnose baker's asthma, occupational sensitization to wheat flour should be distinguished clearly from influences of cosensitization such as it may exist in pollinosis patients. To define the route of sensitization, the cross-reactivity between wheat flour and grass pollen allergens was investigated. METHODS: Two groups of atopic individuals with or without professional contact to wheat flour were screened by skin prick test for their sensitization to wheat flour and grass pollen and, in the case of hints for cosensitization, by Enzyme Allergo Sorbent Test (EAST). Cross-reactivity between wheat flour and grass pollen allergens was investigated by IgE binding inhibition assay using sera of 20 cosensitized individuals and by immunoblots. RESULTS: The immunological cross-inhibition between wheat flour and grass pollen proves some proteins to share common allergenic determinants. Significant differences between bakers and individuals not occupationally exposed to flour could be seen in the inhibition of IgE binding to wheat flour. While the IgE binding to wheat flour allergens was only slightly inhibited by grass pollen proteins in baker's sera its inhibition was nearly complete by this extract in sera of nonexposed atopics. Immunoblots indicate that wheat proteins with a molecular weight of about 8-12 and 22 kD preferentially react with sensitized bakers' IgE and not with IgE of other individuals. IgE binding to grass pollen allergens in immunoblots and inhibition experiments showed no differences between bakers and other subjects. CONCLUSION: In the group of atopics without professional contact to flour, the positive test results from wheat flour obviously ensue from contact to cross-reacting grass pollen proteins whereas bakers are exposed and sensitized to allergens of both sources. Such cross-inhibition experiments help to identify the source of sensitization which may be difficult to obtain in case of cross-reacting allergens.