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.

Spatio-temporal analysis of glass volume processing using ultrashort laser pulses.

Ultrashort laser pulses allow for the in-volume processing of glass through non-linear absorption, resulting in permanent material changes and the generation of internal stress. Across the manifold potential applications of this technology, process optimization requires a detailed understanding of the laser-matter interaction. Of particular relevance are the deposition of energy inside the material and the subsequent relaxation processes. In this paper, we investigate the spatio-temporal evolution of free carriers, energy transfer, and the resulting permanent modifications in the volume of glass during and after exposure to femtosecond and picosecond pulses. For this purpose, we employ time-resolved microscopy in order to obtain shadowgraphic and interferometric images that allow relating the transient distributions to the refractive index change profile. Whereas the plasma generation time is given by the pulse duration, the thermal dynamics occur over several microseconds. Among the most notable features is the emergence of a pressure wave due to the sudden increase of temperature and pressure within the interaction volume. We show how the structure of the modifications, including material disruptions as well as local defects, can be directly influenced by a judicious choice of pulse duration, pulse energy, and focus geometry.

Dental and Temporomandibular Joint Pathology of the Grey Wolf (Canis lupus).

Skulls from 392 grey wolves (Canis lupus) were examined macroscopically according to predefined criteria. Two hundred and seven skulls were included in this study, comprised of 124 young adults (59.9%) and 83 adults (40.1%); of these, 65 (31.4%) specimens were from male wolves and 104 (50.3%) were from females, with 38 (18.4%) of unknown sex. Out of 8,694 possible teeth, 8,339 (95.9%) were present for evaluation. Fifty-five teeth (15.5%) were absent congenitally, 30 (8.5%) were lost during life and 270 (76.1%) were lost artefactually post mortem. Skeletal or dental malocclusion was present in 37 specimens (17.9%), with level bite being the most commonly encountered malocclusion. Enamel hypoplasia was present in five skulls (2.4%), affecting eight teeth (0.1%) in total. An abnormal number of roots was found on 23 teeth (0.3%) on 13 skulls (6.3%). Persistent deciduous teeth occurred in two (1.0%) specimens, affecting one (0.01%) tooth each. Fenestration or dehiscence was found associated with 203 teeth (2.4%) in 72 skulls (34.8%). Periodontitis was noted on 115 skulls (55.6%) and 1,000 teeth (11.5%), affecting significantly more adults (n = 63, 75.0%) than young adults (n = 52, 41.9%; P <0.0001). One hundred and sixty-one skulls (77.8%) showed signs of endodontal disease, including attrition or abrasion on 144 skulls (69.6%) and 2,522 teeth (30.2%) and 424 fractured teeth (5.1%) on 103 skulls (49.8%). Both lesions affected significantly more adults than young adults. Overt periapical disease was associated with six teeth (0.1%) distributed across five skulls (2.4%). A carious lesion was present on one tooth (0.01%) of one specimen (0.5%). Lesions consistent with temporomandibular joint (TMJ) osteoarthritis were found in 24 specimens (11.6%), affecting 38 joints (9.2%). Trauma to the skull, such as bite marks, bullet holes or blunt trauma, was noted in 44 skulls (21.2%). The grey wolf and the domestic dog (Canis lupus familiaris) share common dental diseases; however, the proportion and severity may vary. Although the clinical significance of dental and TMJ pathology in the grey wolf remains unknown, based on the impact of these disorders on the domestic dog, the occurrence and severity of these lesions are likely to play an important role in the morbidity and mortality of this wild canid species.

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.

Dental and Temporomandibular Joint Pathology of the North American Brown Bear (Ursus arctos horribilis, Ursus arctos middendorffi and Ursus arctos sitkensis).

Museum specimens (maxillae and mandibles) from 393 North American brown bears (Ursus arctos) from Alaska were examined macroscopically according to predefined criteria and 204 specimens were included in this study. The specimens were acquired between 1905 and 2012. There were 99 specimens (48.5%) from male animals, 87 (42.7%) from female animals and 18 (8.8%) from animals of unknown sex. The ages of the animals ranged from neonate to adult, with 92 adults (45.1%) and 112 young adults (54.9%) included and neonates/juveniles excluded from the study. The number of teeth available for examination was 6,525 (76.2%); 8.6% of teeth were absent artefactually, 0.8% were deemed absent due to acquired tooth loss and 14.5% were absent congenitally. None of the brown bears had supernumerary teeth, persistent deciduous teeth or abnormally formed crowns. Only four of the specimens in the present population were affected by enamel hypoplasia and one specimen contained two mandibular fourth premolar teeth with one root instead of two. All 204 specimens displayed at least some degree of attrition/abrasion, affecting 63% of all teeth, ranging from mild wear of the enamel to deep abrasion associated with pulp exposure. Ten-times more adult than young adult specimens had abrasion causing pulp exposure, while more young adults showed mild attrition/abrasion. Dental fractures were noted in one-third of brown bears and in 3.0% of the total number of present teeth. More adult brown bears had dental fractures than young adults. There were 11 specimens (5.4%) that displayed overt periapical disease, affecting a total of 20 dental alveoli (0.22%), with adults significantly more affected than young adults. Some degree of periodontitis was seen in 145 specimens (71.1%), affecting 13.6% of all dental alveoli. Nearly one-third (29.9%) of skulls displayed skeletal and/or dental malocclusion, most commonly a level bite. Lesions consistent with temporomandibular joint (TMJ) osteoarthritis were found in 27 specimens (13.2%). Caries lesions were discovered in four specimens (2.0%), affecting eight teeth in total. Although the clinical significance of dental and TMJ pathology in the brown bear remains elusive, the occurrence and severity of some of these lesions may play an important role in the morbidity of this species.

Minor allergen patterns in birch pollen allergen products-A question of pollen?

BACKGROUND: Contrary to the scientific differentiation between major and minor allergens, the regulatory framework controlling allergen products in the EU distinguishes relevant and non-relevant allergens. Given the lack of knowledge on their clinical relevance, minor allergens are usually not controlled by allergen product specifications. Especially, in birch pollen (BP) allergen products, minor allergens are commonly disregarded. OBJECTIVES: To quantify three minor allergens in BP allergen products from different manufacturers and to assess the influence of the utilized BP on minor allergen patterns. METHODS: Apart from common quality parameters such as Bet v 1 content, Bet v 4, Bet v 6 and Bet v 7 were quantified in 70 BP allergen product batches from six manufacturers, using ELISA systems developed in-house. Batch-to-batch variability was checked for agreement with a variability margin of 50%-200% from mean of the given batches for individual allergen content. Subsequently, minor allergen patterns were generated via multidimensional scaling and related to information on the pollen lots used in production of the respective product batches. RESULTS: Like the already established Bet v 4 ELISA, the ELISA systems for quantification of Bet v 6 and Bet v 7 were successfully validated. Differences in minor allergen content between products and batch-to-batch consistency were observed. Correlations between minor and major allergen content were low to moderate. About 20% of batches exceeded the variability margin for at least one minor allergen. Interestingly, these fluctuations could not in all cases be linked to the use of certain BP lots. CONCLUSIONS AND CLINICAL RELEVANCE: The impact of the observed minor allergen variability on safety and efficacy of BP allergen products can currently not be estimated. As the described differences could only in few cases be related to the used pollen lots, it is evident that additional factors influence minor allergens in BP allergen products.

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.

Laser-generated broadband antireflection structures for freeform silicon lenses at terahertz frequencies.

We present a flexible technology to generate broadband antireflection (AR) structures for the terahertz (THz) frequency range on planar and curved surfaces of silicon optics. Ultrashort laser pulses are used to ablate the surface to form a pattern of conical pillars with a period of 30 µm. These subwavelength structures act as an effective medium with gradual transition of the refractive index from air to silicon, which reduces the Fresnel reflection losses. The characterization with the THz time-domain spectroscopy system shows an AR effect for a frequency range of 0.1-1.5 THz with a maximum enhancement of the spectral amplitude by ca. 32% at 0.4 THz for planar surfaces. In addition, we demonstrate laser-generated AR structures on convex silicon lenses of both photoconductive emitter and detector devices. Here, the THz pulse amplitude can be increased by about 28%, and single frequencies even show an improvement of the spectral amplitude up to 58%.

A longitudinal study to characterize the distribution patterns of Mycobacterium avium ssp. paratuberculosis in semen, blood and faeces of a naturally infected bull by IS 900 semi-nested and quantitative real-time PCR.

Johne's disease is caused by Mycobacterium avium ssp. paratuberculosis (MAP) and has been recognized as an important bacterial infection in ruminants. Although MAP has been detected in semen and within the reproductive organs of bulls, the bacterial distribution and shedding patterns are currently not well characterized. Our investigation was performed to detect and quantify MAP in faeces, semen and blood samples repeatedly drawn from a naturally infected but asymptomatic 18-month-old German Simmental breeding bull candidate over a period of 3 years (June 2007-November 2010). Qualitative and quantitative polymerase chain reaction (PCR) techniques were used to correlate the presence and matrix-specific amounts of MAP. In total, 65 sampling dates were selected. Mycobacterium avium ssp. paratuberculosis was detected intermittently in all matrices with MAP-free intervals of up to 18 weeks by an IS900 semi-nested PCR. The number of MAP-positive results from semen and blood samples was higher than from faecal samples. A quantitative polymerase chain reaction detected the highest MAP contents in faeces (10(3) -10(6) MAP/g), while lower amounts were found in semen and blood samples (10(2) -10(5) MAP/ml). Although no significant agreement was calculated between the presence of MAP in faeces and blood, a statistically significant positive correlation between its occurrence in semen and blood was determined (r = 0.38, P < 0.05, n = 29). The present study contributes to a more detailed understanding of MAP distribution patterns in faeces, semen and blood of a subclinically infected breeding bull candidate. It highlights the possible role of breeding bulls as a source of MAP transmission and indicates the need for further monitoring and hygienic measures to prevent the spread of the infection via semen.

Femtosecond and picosecond laser drilling of metals at high repetition rates and average powers.

The influence of pulse duration on the laser drilling of metals at repetition rates of up to 1 MHz and average powers of up to 70 W has been experimentally investigated using an ytterbium-doped-fiber chirped-pulse amplification system with pulses from 800 fs to 19 ps. At a few hundred kilohertz particle shielding causes an increase in the number of pulses for breakthrough, depending on the pulse energy and duration. At higher repetition rates, the heat accumulation effect overbalances particle shielding, but significant melt ejection affects the hole quality. Using femtosecond pulses, heat accumulation starts at higher repetition rates, and the ablation efficiency is higher compared with picosecond pulses.

[Comparison of the sonographic detection of experimentally induced squamous cell carcinoma compared with the histological image]. / Vergleich von Malignitätskriterien in B-Scan- und Farbduplexsonographie mit dem histologischen Erscheinungsbild am Beispiel des experimentell induzierten Plattenepithelkarzinoms.

BACKGROUND: Malignant tumours of the head and the neck are characterised by typical signs of malignancy in greyscale- and colour-coded sonography. Sometimes, such criteria cannot be verified, and in such cases it remains unclear whether typical changes do not exist or whether we just cannot detect them with our high-end ultrasound units. We therefore compared our sonographical findings with the histology obtained in experimentally induced tumours. METHODS: Experimental squamous cell carcinoma was induced subcutaneously in nude mice (n = 18), using four different cell lines. Ultrasound examination of the tumours was performed after 98 and 112 days, respectively, Central necrosis, rupture of the capsule as well as the presence of central and peripheral blood vessels were documented before the tumours were excised. They were then sliced in the same direction as sonography had been carried out before. Blood vessels were stained using CD31. RESULTS AND DISCUSSION: 16 tumours grew subcutaneously. In ultrasound imaging, the capsule was always intact, and subcapsular vessels were detected in every tumour. Central necrosis was seen in 10/16 tumours by sonography, but in 15/16 tumours on histological examination. The failure of sonography in detecting necrosis did not correlate with the size of the tumour. Centrally located vessels were found in 12/16 tumours using colour-coded sonography. Histology, however, showed their presence in all 16 tumours. Interestingly, the failure of sonography in detecting vessels did not correlate with tumour size. It seemed that the detection of such vessels was particularly difficult in those tumours which grew very fast. This finding explains why we often find such vessels in small tumours but not in large specimens, where they would normally be expected.

[Variability of coping strategies in coronary artery bypass surgery patients]. / Zur Variabilität des Coping bei Patienten mit aortokoronarer Bypass-Operation.

The state-versus-trait discussion in coping research would become more meaningful if attention is paid to the variability both of coping patterns in individual patients as well as stability of single coping strategies over time. 35 patients undergoing coronary artery bypass surgery were interviewed three times about their coping and stress experience: after cardiac catheterization, on the day before surgery, and six days after surgery. Anxiety and depression were measured. A coping attitude of "positive passivity" was present at all three points of time. While the group means were stable, vast interindividual differences occurred. Also, there was a high degree of scatter in the stability of single coping items; emotion related coping modes were more stable than cognition and action related ones. The variability of the patients' coping patterns correlated positively with the amount of stress experienced and with preoperative depression. Different coping strategies are linked to a different degree with personality traits, emotional coping modes revealing the closest connection. The variability of individual coping efforts might be linked to a personality disposition characterized by a vulnerability for stress and depressive reactions.

Analysis of phytochelatins in plant matrices by pre-column derivatization, high-performance liquid chromatography and fluorescence-detection.

A sensitive method for the determination of phytochelatins in plant matrices by pre-column derivatization with monobromobimane (mBrB) and high-performance liquid chromatography (HPLC) on reversed phases and fluorescence-detection has been developed and applied to cucumber sprouts (Cucumis sativus) treated with cadmium and to the water moss Fontinalis antipyretica (Cd in environmentally-relevant concentrations). Whereas phytochelatins were found in the Cd-treated sprouts, no phytochelatins were detected in Fontinalis anitipyretica.

The significance of bilateral EEG abnormalities before and after hemispherectomy in children with unilateral major hemisphere lesions.

The rate of seizure relief following hemispherectomy varies between 50 and more than 80%. There has been particular debate concerning the significance of bilateral electroencephalography (EEG) abnormalities in influencing prognosis. This study was set out to determine the frequency of bilateral EEG abnormalities and their relationship to underlying pathology and outcome. We investigated 28 children with unilateral hemisphere lesions, who underwent hemispherectomy. Interictal and ictal EEGs before and after hemispherectomy were reviewed. Post-operative outcome with respect to seizures was noted. Bilateral EEG abnormalities were seen in 75%, but were more common in children with malformations of cortical development than in patients with acquired cerebral lesions, and were found more often in interictal than in ictal records. Post-operative EEG abnormalities were variable and did not consistently predict outcome. Short-term outcome was similar, irrespective of aetiology. With longer term follow-up, only 47% of children with developmental abnormalities were still seizure-free in contrast to 77% of children with acquired abnormalities. Although the incidence of bilateral EEG abnormalities in patients with major unilateral hemisphere lesions is high, these findings alone should not preclude further consideration for hemispherectomy. Our findings emphasise that the aetiology of the lesion plays a major role in determining outcome.