Standardization of allergen products: 3. Validation of candidate European Pharmacopoeia standard methods for quantification of major birch allergen Bet v 1.

BACKGROUND: The BSP090 project aims at establishing European Pharmacopoeia Reference Substances in combination with the corresponding ELISA methods for the quantification of major allergens in allergen products. Two sandwich ELISAs proved suitable for quantification of Bet v 1, the major birch pollen allergen, in preceding phases of BSP090. METHODS: Two Bet v 1-specific ELISA systems were compared with respect to accuracy and precision in a ring trial including 13 laboratories. Model samples containing recombinant rBet v 1.0101 as well as native birch pollen extracts were measured independently at least three times in each facility. The assessment was completed with a comparative quantification of Bet v 1 in 30 marketed birch allergen products in one laboratory, simulating the future use as reference method. RESULTS: In the collaborative study, both candidate ELISAs confirmed their suitability to quantify recombinant and native Bet v 1. ELISA-A showed higher precision and lower interlaboratory variability, yet ELISA-B exhibited slightly higher accuracy. Subsequent parallel measurement of Bet v 1 in a panel of 'real-life' birch allergen products indicated better repeatability of ELISA-B. Both systems detected substantial differences in Bet v 1 content between allergen products, but the effect was more pronounced using ELISA-B due to persistently higher values compared to ELISA-A. CONCLUSIONS: In the collaborative study, no deciding differences were observed between the two candidate ELISAs. Further comparison under conditions simulating the intended use combined with the criterion of long-term availability enabled the selection of one Bet v 1-specific ELISA for proposal as European Pharmacopoeia standard method.

Vaccination with recombinant modified vaccinia virus Ankara prevents the onset of intestinal allergy in mice.

BACKGROUND: Modified vaccinia virus Ankara (MVA)-encoding antigens are considered as safe vaccine candidates for various infectious diseases in humans. Here, we investigated the immune-modulating properties of MVA-encoding ovalbumin (MVA-OVA) on the allergen-specific immune response. METHODS: The immune-modulating properties of MVA-OVA were investigated using GM-CSF-differentiated BMDCs from C57BL/6 mice. OVA expression upon MVA-OVA infection of BMDCs was monitored. Activation and maturation markers on viable MVA-OVA-infected mDCs were analyzed by flow cytometry. Secretion of INF-γ, IL-2, and IL-10 was determined in a co-culture of BMDCs infected with wtMVA or MVA-OVA and OVA-specific OT-I CD8(+) and OT-II CD4(+ ) T cells. BALB/c mice were vaccinated with wtMVA, MVA-OVA, or PBS, sensitized to OVA/alum and challenged with a diet containing chicken egg white. OVA-specific IgE, IgG1, and IgG2a and cytokine secretion from mesenteric lymph node (MLN) cells were analyzed. Body weight, body temperature, food uptake, intestinal inflammation, and health condition of mice were monitored. RESULTS: Infection with wtMVA and MVA-OVA induced comparable activation of mDCs. MVA-OVA-infected BMDCs expressed OVA and induced enhanced IFN-γ and IL-2 secretion from OVA-specific CD8(+ ) T cells in comparison with OVA, wtMVA, or OVA plus wtMVA. Prophylactic vaccination with MVA-OVA significantly repressed OVA-specific IgE, whereas OVA-specific IgG2a was induced. MVA-OVA vaccination suppressed TH 2 cytokine production in MLN cells and prevented the onset of allergic symptoms and inflammation in a mouse model of OVA-induced intestinal allergy. CONCLUSION: Modified vaccinia virus Ankara-ovalbumin (MVA-OVA) vaccination induces a strong OVA-specific TH 1- immune response, likely mediated by the induction of IFN-γ and IgG2a. Finally, MVA-based vaccines need to be evaluated for their therapeutic potential in established allergy models.

Airborne olive pollen counts are not representative of exposure to the major olive allergen Ole e 1.

Pollen is routinely monitored, but it is unknown whether pollen counts represent allergen exposure. We therefore simultaneously determined olive pollen and Ole e 1 in ambient air in Córdoba, Spain, and Évora, Portugal, using Hirst-type traps for pollen and high-volume cascade impactors for allergen. Pollen from different days released 12-fold different amounts of Ole e 1 per pollen (both locations P < 0.001). Average allergen release from pollen (pollen potency) was much higher in Córdoba (3.9 pg Ole e 1/pollen) than in Évora (0.8 pg Ole e 1/pollen, P = 0.004). Indeed, yearly olive pollen counts in Córdoba were 2.4 times higher than in Évora, but Ole e 1 concentrations were 7.6 times higher. When modeling the origin of the pollen, >40% of Ole e 1 exposure in Évora was explained by high-potency pollen originating from the south of Spain. Thus, olive pollen can vary substantially in allergen release, even though they are morphologically identical.

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.

Transanal evisceration of small bowel in two patients with chronic rectal prolapse: case presentation and literature review.

There are fewer than 100 documented cases of transanal small bowel evisceration in the literature. We report two cases of this rare surgical emergency in an 84-year old man and a 79-year old woman. Both patients required urgent laparotomy, resection of ischaemic bowel and transabdominal resection of the rectal defect with colostomy. Postoperative recovery was uneventful. Rare imaging and clinical photography are shared to highlight the extreme nature of this condition. We identified 38 relevant cases of reported bowel evisceration through our literature review. Most patients were elderly women with untreated rectal prolapse. Gynaecological comorbidity was another risk factor. The aetiological mechanism is suspected to stem from chronic ischaemic insult to the rectal wall, resulting in thinning and subsequent perforation. Surgical management may consist of primary suture repair of the rectal tear, or a Hartmann's procedure. Timely intervention is essential to minimise patient morbidity and mortality.

The non-specific lipid transfer protein, Ara h 9, is an important allergen in peanut.

BACKGROUND: Plant food allergy in the Mediterranean area is mainly caused by non-specific lipid transfer proteins (nsLTP). The aim of this study was to characterize peanut nsLTP in comparison with peach nsLTP, Pru p 3, and assess its importance in peanut allergy. METHODS: Peanut-allergic patients from Spain (n=32) were included on the basis of a positive case history and either a positive skin prick test or specific IgE to peanut. For comparison, sera of 41 peanut-allergic subjects from outside the Mediterranean area were used. Natural Ara h 9 and two isoforms of recombinant Ara h 9, expressed in Pichia pastoris, were purified using a two-step chromatographic procedure. Allergen characterization was carried out by N-terminal sequencing, circular dichroism (CD) spectroscopy, immunoblotting, IgE inhibition tests and basophil histamine release assays. RESULTS: Compared with natural peanut nsLTP, the recombinant proteins could be purified in high amounts from yeast supernatant (> or =45 mg/L). The identity of the proteins was verified by N-terminal amino acid sequencing and with rabbit nsLTP-specific antibodies. CD spectroscopy revealed similar secondary structures for all preparations and Pru p 3. The Ara h 9 isoforms showed 62-68% amino acid sequence identity with Pru p 3. IgE antibody reactivity to rAra h 9 was present in 29/32 Spanish and 6/41 non-Mediterranean subjects. Recombinant Ara h 9 showed strong cross-reactivity to nPru p 3 and similar IgE-binding capacity as nAra h 9. The two Ara h 9 isoforms displayed similar IgE reactivity. In peanut-allergic patients with concomitant peach allergy, Ara h 9 showed a weaker allergenic potency than Pru p 3 in histamine release assays. CONCLUSIONS: Ara h 9 is a major allergen in peanut-allergic patients from the Mediterranean area. Ara h 9 is capable of inducing histamine release from basophils, but to a lesser extent than Pru p 3.

Validation of ELISA methods for quantification of the major birch allergen Bet v 1 (BSP090).

To date, the potency of allergen products in Europe is expressed in manufacturer-specific units relative to a product-specific in-house reference. Consequently, cross-product comparability of allergen products from different manufacturers with respect to strength and efficacy is impossible. The Biological Standardisation Programme (BSP) project BSP090 addresses this issue via the establishment of reference standards in conjunction with ELISA methods for the quantification of major allergens in allergen products. Since the initiation of BSP090, the recombinant major allergen Bet v 1 has been adopted by the European Pharmacopoeia Commission as a Chemical Reference Substance (CRS). In parallel, two sandwich ELISA systems for quantification of Bet v 1 were found suitable in preliminary phases of BSP090 to be validated in a large collaborative study. In this study, the candidate ELISA systems were compared with respect to accuracy, precision and variability. Thirteen participating laboratories tested model samples containing the CRS as well as spiked and unspiked birch pollen extracts. Both in pre-testing and in the collaborative study, the 2 candidate ELISA systems confirmed their suitability to quantify recombinant and native Bet v 1. As no clear-cut decision for one of the ELISA systems could be made based on the results of the collaborative study, a post-study testing was performed. Bet v 1 content of 30 birch pollen allergen products was determined in parallel in both ELISA systems. Consequently, 1 candidate ELISA system was selected to be proposed as the future European Pharmacopoeia standard method for Bet v 1 quantification.

Dormont High School (Pittsburgh, Pennsylvania) blood pressure study.

A cohort of high school students examined in a school health study between 1957-63 were followed to 1977, when as adults still living in the area, 373 (71%) were reexamined. Mean age at 17-year follow-up was 34 years. The mean systolic blood pressure (SBP) at follow-up for men was 125 mm Hg, women 111 mm Hg. The boys' SBP had increased 4.0 Hg while the girls' declined 4.0 mm Hg in 17 years. The boys had gained an average of 37.2 lbs, and 1 inch, the girls 16.7 lbs, and 0.5 inch. Tracking was studied in several ways. The correlation coefficient of the SBP taken 17 years apart ws 0.44 for boys and 0.39 for girls. Current SBP was 115 mm Hg for boys with the lowest tenth of high school SBPs and 131 mm Hg for the boys in the highest tenth. Thirty-nine had hypertension, DBP greater than or equal to 90, or were on antihypertensive medication. They had had substantially higher SBP and weight in high school, and had gained more weight from high school to adult life than controls. After adjusting for high school SBP, weight gain for boys was the major determinant of subsequent high blood pressure (BP).

Enhancement of murine IgE antibody detection by IgG removal.

RATIONALE: Although animal models for the study of allergic reactions are desirable, the use of mice has been hindered by the lack of sufficiently sensitive in vitro immunoglobulin epsilon (IgE) antibody assays. The aim of this study was to enhance IgE antibody measurements by immunoglobulin gamma (IgG) depletion. METHODS: Seven- to eight-week-old female mice of four strains (C3H/HeJ, CBA/J, C57Bl/6J, and Balb/c) were immunized (20 mice/group) with shrimp or peanut extracts using Al(OH)(3) as adjuvant. Following immunization, animals were sacrificed by exsanguination and the sera of each group pooled. Initial measurements of IgE antibody levels by enzyme-linked immunosorbent assay (ELISA) were relatively low; IgG and IgE reactivity patterns by immunoblot were similar. Thus, sera from shrimp or peanut immunized mice were depleted of IgG (absorbed 3-6 times with immobilized protein G) and then tested for IgE antibody to shrimp or peanut allergen. RESULTS: A 3- to 5-fold increase in IgE antibody reactivity as measured by ELISA was demonstrated when >80-90% of the IgG was removed. This increase in detection of allergen-specific IgE occurred in sera from all mouse strains and to all allergens tested. In addition, reactivity of IgE antibodies to peanut or shrimp allergens by immunoblot increased visually approximately 4- to 10-fold. CONCLUSIONS: These studies indicate that allergen-specific IgG antibodies, which may be in more than 100-fold excess to IgE antibodies, interferes with detection of allergen-specific IgE, probably by competitive binding to allergenic epitopes. Substantial depletion of IgG antibodies (>80%) result in a significant increase in the sensitivity of the antibody measurements.

Target detection against narrow band noise backgrounds.

We studied the detectability of narrow band random noise targets embedded in narrow band random noise backgrounds as a function of differences in center frequency, spatial frequency bandwidth and orientation bandwidth between target and the immediately adjacent background. Unlike most target detection experiments the targets were not added to the background; they replaced the underlying background texture. Simulations showed that target detection probabilities could be accounted for by a simple transformation on the summed outputs of a two layer filter model similar to the complex channels model proposed by Graham, Beck and Sutter (Graham, N., Beck, J., & Sutter, A. (1992). Vision Research, 32, 719-743). Subsequently, the model was tested on the detection of camouflaged vehicle targets with encouraging results.

Seafood allergy and allergens: a review.

Seafoods are composed of diverse sea organisms and humans are allergic to many of them. Tropomyosin is a major allergen in many shellfish, especially crustacea and mollusks. Interestingly, tropomyosin has also been identified as an important allergen in other invertebrates including dust mites and cockroaches, and it has been proposed by some to be an invertebrate pan allergen. Different regions of shrimp tropomyosin bind IgE; 5 major IgE-binding regions have been identified in shrimp tropomyosin containing 8 epitopes. Mutations of these shrimp allergenic epitopes can reduce seafood allergenicity; methods utilizing such mutations will provide safer vaccines for more effective treatment of seafood-allergic patients, and in the future less-allergenic seafood products for consumption.

IgE and monoclonal antibody reactivities to the major shrimp allergen Pen a 1 (tropomyosin) and vertebrate tropomyosins.

Pen a 1, the major shrimp allergen from brown shrimp Penaeus aztecus has been identified as the muscle protein tropomyosin. To identify Pen a 1 IgE binding sites, the reactivities of Pen a 1-specific monoclonal antibodies (mAbs) and shrimp-allergic subjects' IgE to shrimp and homologous mammalian tropomyosins were analyzed. Pen a 1, purified by preparative SDS-PAGE and commercially obtained porcine, bovine and rabbit tropomyosin were cleaved by CNBr or digested by endoproteinases Lys-C, Glu-C, trypsin, Arg-C and chymotrypsin. Reactivities of Pen a 1-specific mAbs and IgE to the resulting peptides were analyzed by dot blot and immunoblotting. The dot blot analysis showed that mAbs and IgE antibodies did not react with any of the mammalian tropomyosins. The immunoblot analysis showed that all Pen a 1 digests bound IgE or mAbs. However, not all peptides in each digest possessed an IgE binding site. IgE binding intensity and frequency varied by subject and peptide digest. IgE and mAb reactivity patterns were similar but no mAb reproduced the IgE binding patterns indicating that subject' IgE bound some epitopes that were not recognized by the Pen a 1-specific mAbs. These studies suggest that IgE-binding epitopes are restricted to certain parts of the Pen a 1 molecule, Pen a 1 may have several similar epitopes, and that Pen a 1 epitopes do not appear to be located in the highly homologous parts of the tropomyosin molecule.

The Poisson process as a model for compartment digesta flow in ruminants.

The Poisson process, the simplest stochastic flow process, was used to develop a multicompartment model of ruminant digesta flow with Gamma distributed retention times. Although mathematically the model is a generalization of many previously published models, the physiological model differs substantially in asserting that the distributed delay time and the exponential rate (scale) parameters, including the scale parameter of the Gamma distribution, are determined by total digesta flow, and thus invariant with respect to the fraction marked. The shape factor of the Gamma distribution is shown to be sufficient to explain the difference between markers in rate of marker excretion. Consequently, the parameters of multiple markers can be simultaneously estimated with the constraint that the exponential scale parameters and the delay time are invariant with respect to marker. This constraint leads to a measure of pure error to strengthen statistical tests for model rejection. Steady-state digesta retention time is estimated from the transient marker retention parameters, eliminating the necessity of speculating on what fraction of digesta the marked fraction represents. Tests of various models, using simulations and animal experiments indicate that, even if a model is correct, it is not possible to obtain reliable parameter estimates by fitting to a single marker. Even with multiple markers some caution must be used in interpreting parameter estimates derived from least squares fitting.

Effects of energy supplementation on lamb production of Javanese thin-tail ewes.

A 3-yr study was conducted in North Sumatra, Indonesia, as part of an evaluation of the feasibility of integrating sheep and rubber production. The objective was to evaluate the effects of increasing energy supplementation on reproduction and other performance criteria of Javanese Thin-Tail sheep grazing volunteer forages under 8- and 14-yr-old rubber trees. The control group was unsupplemented. The medium group was supplemented with high-energy feeds at 1% of the flock body weight, with the low and high groups receiving 60% or 140% of the daily energy provided by the medium group diet. Supplements provided 1.2 g protein per kilogram BW. There were 158 lambs born to the 152 ewes in the 1st year of the study. Preweaning mortality rates of lambs were reduced (P less than .01) with supplementation (45 vs 12, 3 and 12% for the control, low, medium and high groups, respectively). During the 3 yr, litter size was higher (P less than .01) in the high group (1.33, 1.31 and 1.34 vs 1.71 for ewes on the four respective diets). Observed repeatability of litter size of individual ewes in all treatment groups for the first three parities was higher (P less than .01) than would be expected if litter size were a random event. Of the lambs born in the 1st yr, kilograms of lamb weaned per ewe joined were 3.1, 7.8, 7.3 and 9.8. At prevailing prices, only the high supplement level was profitable compared to the control. For the high group, the added return from the sale of lambs born the 1st yr was 120% of the added cost of supplementing the ewes until all the lambs were weaned (15 mo). Response of sheep to the high level of energy supplementation, especially with regard to litter size, indicates that supplementing sheep grazing in rubber plantations at a high level can be profitable.

The use of blends of cassava flour and extruded full-fat soybeans in diets for broiler chickens.

A study was conducted to determine the effects of blending different levels of a low-prussic acid cassava flour with extruded full-fat soybeans in diets for growing broiler chickens. The full-fat soybeans contribute oil which increases the energy content of the diet, aids in overcoming the dusty nature of cassava, and provide high-quality protein. One-third, two-thirds, and all of the maize was replaced by cassava in diets with none, 12.5 and 25% extruded full-fat soybeans. Diets were fed in pelleted form to broiler chickens for a 47-day feeding trial. Replacement of one-third of the maize with cassava had no adverse effects on body weight gains in this study with a reduction in weight at higher levels at the conclusion of the study. Feed utilization was reduced more severely than was anticipated. However, growth rate on the higher levels of cassava was reasonably good, indicating that producers might feed these diets for a slightly longer period of time and produce chickens more economically if cassava meal were available at a cost significantly less than that of maize.

Establishment of recombinant major allergens Bet v 1 and Phl p 5a as Ph. Eur. reference standards and validation of ELISA methods for their measurement. Results from feasibility studies.

The potency of allergen extracts is determined as total allergenic activity without consideration of their composition and the units differ from one manufacturer to another, making it very difficult to compare the different products. Recently, purified major allergens have been obtained by recombinant DNA technology and produced under Good Manufacturing Practice (GMP) conditions. In principle, such recombinant allergens could be established as reference standards and could help for the standardisation of the major allergen content of allergen extracts. Two recombinant major allergens, one from birch pollen, rBet v 1, and one from Timothy grass pollen, Phl p 5a, have been selected at the end of the CREATE programme as a potential starting point for the establishment as European Pharmacopoeia (Ph. Eur.) Reference Standards through a project run by the Biological Standardisation Programme (BSP) of the European Directorate for the Quality of Medicines & HealthCare (EDQM). To this end, bulk candidate recombinant materials, produced under GMP conditions, were procured from two European manufacturers and subsequently formulated and lyophilised. Four ELISA systems from three different manufacturers were included in the project, two for Bet v 1 and two for Phl p 5a with the aim of establishing reference methods for determination of the respective major antigens both in natural allergen extracts as well as in recombinant allergen products. The project was run in 3 phases: a preparatory and preliminary testing phase (feasibility phase or Phase 1), an extended feasibility phase carried out in 3 laboratories (Phase 2) to confirm the transferability of the methods and an international collaborative study with a large number of participating laboratories (Phase 3). This article describes the work done in Phase 1 and Phase 2, i.e. the physico-chemical and biological characterisation of the recombinant candidate reference standards, the assessment of their suitability for the intended purpose as well as the evaluation of the candidate ELISA systems. The results show that both candidate reference standards are suitable for the intended purpose. In addition, three out of the four ELISA systems that were included in the preliminary phase were found to be appropriate for further evaluation in the collaborative study which was organised in 2011. The results of the collaborative study will be published separately.