The major grass pollen group 5 allergen from Dactylis glomerata and its C-terminal split product both behave as dimers: implications for allergen standardization

BACKGROUND: On SDS-PAGE grass pollen group-5 allergens migrate as a doublet with an apparent molecular mass (M(r)) of 25 kDa. Immunoblot analysis revealed additional group 5 reactivity at double and half this M(r). The aim of this study was to investigate these group 5 molecular entities and to compare their allergenicity and behavior in quantitative immunoassays. METHODS: Group-5-specific monoclonal antibodies were produced and used for the development of a group-5-specific sandwich ELISA. Affinity-purified Dac g 5 was separated by SDS-PAGE/Western blotting; individual bands were analyzed by N-terminal sequencing. Size exclusion chromatography (SEC) in conjunction with group-5-specific ELISA, competitive RIA and RAST inhibition were used to analyze the size distribution of Dac g 5. Basophil histamine release assays were used to assess biological activity. RESULTS: The lower band of the typical group 5 doublet was identified as a truncated form lacking the typical group 5 N-terminus AD(L)/(A)GY, observed in the upper band. The 12-kDa peptide was shown to be the C-terminal half of Dac g 5 (amino acid 127 onwards). SEC in conjunction with competitive RIA revealed that around 45% of Dac g 5 is represented by the 12-kDa peptide. Both the C-terminal half and the whole allergen dimerize under nondenaturing conditions. In competitive RIA and RAST inhibition both forms are equally well detected. In contrast, the half molecule is poorly recognized in sandwich ELISA and displays negligible biological activity in basophil histamine release tests with purified IgE. CONCLUSIONS: These observations stress the need to evaluate the performance of allergen standardization protocols in detail, with special attention to allergen size distribution.     

Genetic counseling for familial conditions during pregnancy: an analysis of patient characteristics

Reproductive genetic counseling for a familial genetic risk factor preferably takes place before conception. However, of the women with a family history of genetic conditions who attend our department of clinical genetics, about 10-20% attend for the first time during a pregnancy. The current study aims to explore patient-related factors that may affect this late timing of reproductive genetic counseling. Consecutive pregnant (n = 100) and non-pregnant (n = 84) women visiting the department of clinical genetics for a genetic risk factor which was not age related completed a questionnaire immediately prior to the consultation. The questionnaire asked for (a) background characteristics, i.e. socio-demographic, obstetric, and disease characteristics (b) cognitive factors, i.e. initiative of referral, knowledge of the risk factor involved, risk perception, worry, child wish, attitudes toward abortion, and preferred participation in decision making, and (c) reasons for the timing of the consultation and for seeking genetic counseling. Pregnant women appeared to be higher educated, considered their children more often as healthy and were less often affected themselves, as compared to non-pregnant women. They also estimated their chance of having an affected child as lower, and they worried less. Furthermore, the initiative for referral was taken less often by the pregnant woman herself and more often by a medical worker. There were no major differences between the two study groups in knowledge, perceived severity of the risk factor, child wish, attitudes toward abortion, desired participation in decision making, and reasons to seek genetic counseling. Women indicated no specific reasons for their timing of referral for reproductive genetic counseling, e.g. during vs before pregnancy. Our data suggest that this timing of referral is not influenced predominantly by the women's level of knowledge. Rather, women's estimation of genetic risks and their degree of worry, which may be in accordance with the actual risk figures, seem to play a role in seeking genetic counseling. Although further studies are required, a more active role of health care providers seems warranted if we want to prevent genetic counseling for familial genetic conditions during pregnancy as much as possible.     

Characterization of natural Dac g 1 variants: an alternative to recombinant group 1 allergens

BACKGROUND: Production of soluble correctly folded recombinant group 1 allergens has proven to be difficult. Purified natural group 1 allergens could be an alternative for application in immunotherapy. OBJECTIVE: Cloning and expression of recombinant Dac g 1; purification of natural Dac g 1 variants and immunochemical characterization of these molecules. METHODS: Dac g 1 was cloned and expressed in the yeast Pichia pastoris. Hydrophobic interaction (HIC), size exclusion, and/or affinity chromatography were used to purify Dac g 1 from Dactylis glomerata pollen extract. Dac g 1 variants were analyzed by N-terminal sequencing. Immune reactivity was assessed by sandwich ELISA, competitive RIA, RAST (inhibition), and in vitro basophil histamine release tests. RESULTS: Dac g 1 was cloned, revealing up to 98% amino acid sequence homology to other group 1 allergens. Purification of natural Dac g 1 revealed at least 3 variants, with an apparent molecular mass (Mr) on SDS-PAGE of 33 kd (HMr), 30 kd (IMr) and 28 kd (LMr). Extraction of IMr Dac g 1 required 0.9% saline, whereas the other 2 variants were also extractable in water. The N-terminus of HMr and IMr Dac g 1 differs at 2 positions, and LMr Dac g 1 was shown to be N-terminally truncated, lacking the first 30 amino acids. The nonretarded fraction of HIC commonly used in group 1 purification protocols does not contain this LMr molecule. IMr Dac g 1 was poorly recognized in 2 of 3 sandwich ELISAs and competitive RIA but demonstrated similar biological activity compared with HMr Dac g 1. CONCLUSIONS: Natural Dac g 1 variants can be separated by extraction of pollen in the presence or absence of saline followed by HIC and size exclusion chromatography. Thus, purified Dac g 1 is an alternative to recombinant group 1 allergens.     

Quality of life after brachytherapy in patients with glioblastoma multiforme

As quality of life (QoL) is perhaps the most important outcome for patients treated for glioblastoma multiforme (GBM), we measured QoL in GBM patients after brachytherapy. QoL was assessed by questionnaires for both patients and partners before brachytherapy and at various times during follow-up in 21 GBM patients by an extension of the Rotterdam Symptom Checklist (e-RSCL), consisting of four subscales. The Karnofsky Performance Scale (KPS) was also measured. Analysis of variance was done to evaluate the direct effect of brachytherapy (visit 1-2, short-term) and during follow up (visit 1-4, longer-term). Significant short-term effects were found for two subscales of the e-RSCL. Longer-term effects were found for all four subscales and for the KPS. A high correlation between partner and patient's QoL assessment was found. QoL in GBM patients after brachytherapy can therefore be carefully monitored with a subjective instrument such as the e-RSCL. Patients and partners experience QoL equally.     

Costs of quality management systems in long-term care organizations: an exploration

The article describes a method for measuring and reporting the costs of quality management in 11 long-term care organizations (nursing homes, home health care organizations, and homes for the elderly) and a national survey in 489 organizations providing long-term care. Site visits and a questionnaire were used to measure the existence of quality management (QM) activities and investigate the costs per QM activity in more detail. Health care organizations differentiate between regular activities and QM activities. The costs of QM activities were found to vary between 0.3% and 3.5% of the budget in three nursing homes. An extrapolation of the costs of QM activities to the entire sector shows that the long-term care sector spent between 0.8% and 3.5% of the overall budget for QM in 1999. The costs of developing and implementing QM activities are higher than the costs of monitoring. Most long-term care organizations have no insight into failure costs (i.e. the costs of quality deviations). This makes it impossible for health care organizations to draw conclusions about the cost-effectiveness of QM.