Suggestions for the assessment of the allergenic potential of genetically modified organisms

The prevalence of allergic diseases has been increasing continuously and, accordingly, there is a great desire to evaluate the allergenic potential of components in our daily environment (e.g., food). Although there is almost no scientific evidence that genetically modified organisms (GMOs) exhibit increased allergenicity compared with the corresponding wild type significant concerns have been raised regarding this matter. In principle, it is possible that the allergenic potential of GMOs may be increased due to the introduction of potential foreign allergens, to potentially upregulated expression of allergenic components caused by the modification of the wild type organism or to different means of exposure. According to the current practice, the proteins to be introduced into a GMO are evaluated for their physiochemical properties, sequence homology with known allergens and occasionally regarding their allergenic activity. We discuss why these current rules and procedures cannot predict or exclude the allergenicity of a given GMO with certainty. As an alternative we suggest to improve the current evaluation by an experimental comparison of the wild-type organism with the whole GMO regarding their potential to elicit reactions in allergic individuals and to induce de novo sensitizations. We also recommend that the suggested assessment procedures be equally applied to GMOs as well as to natural cultivars in order to establish effective measures for allergy prevention.     

Recombinant allergens promote expression of CD203c on basophils in sensitized individuals

BACKGROUND: Traditionally, the diagnosis of type I allergies is based on clinical data, skin test results, and laboratory test results with allergen extracts. During the past few years, several attempts have been made to refine diagnostic assays in clinical allergy by introducing recombinant allergens and novel markers of IgE-dependent cell activation. OBJECTIVES: We have identified the ectoenzyme CD203c as a novel basophil antigen that is upregulated on IgE receptor cross-linkage. In this study we applied CD203c and a panel of recombinant allergens to establish a novel basophil test that allows for a reliable quantification of IgE-dependent responses at the effector cell level. METHODS: Patients allergic to birch (Bet v 1, n = 15; Bet v 2, n = 8) and grass (Phl p 1, n = 15; Phl p 2, n = 10; Phl p 5, n = 14) pollen allergens, as well as 10 nonallergic donors, were examined. Basophils were exposed to various concentrations of recombinant allergens for 15 minutes and then examined for expression of CD203c by means of flow cytometry. CD203c upregulation was correlated with the increase in CD63. RESULTS: Exposure to recombinant allergens resulted in a dose-dependent increase in expression of CD203c on peripheral blood basophils in sensitized individuals, whereas no increase was seen in healthy control subjects. The effects of the recombinant allergens on CD203c expression were also time dependent. There was a good correlation between allergen-induced upregulation of CD203c and upregulation of CD63 (R = 0.76). CONCLUSION: Flow cytometric quantitation of CD203c on blood basophils exposed to recombinant allergens is a useful approach to determine the allergic state in sensitized individuals and represents a basis for a sensitive novel allergy test.     

Effects of topography and composition of titanium surface oxides on osteoblast responses

To investigate the roles of composition and characteristics of titanium surface oxides in cellular behaviour of osteoblasts, the surface oxides of titanium were modified in composition and topography by anodic oxidation in two kinds of electrolytes, (a) 0.2 M H(3)PO(4), and (b) 0.03 M calcium glycerophosphate (Ca-GP) and 0.15 M calcium acetate (CA), respectively. Phosphorus (P: ca.10at%) or both calcium (Ca: 1-6at%) and phosphorus (P: 3-6at%) were incorporated into the anodized surfaces in the form of phosphate and calcium phosphate. Surface roughness was slightly decreased or enhanced (R(a) in the range of 0.1-0.5 microm) on the anodized surfaces. The geometry of the micro-pores in the anodized surfaces varied with diameters up to 0.5 microm in 0.2 M H(3)PO(4) and to 2 microm in 0.03 M Ca-GP and 0.15 M CA, depending on voltages and electrolyte. Contact angles of all the anodic oxides were in the range of 60-90 degrees. Cell culture experiments demonstrated absence of cytotoxicity and an increase of osteoblast adhesion and proliferation by the anodic oxides. Cells on the surfaces with micro-pores showed an irregular and polygonal growth and more lamellipodia, while osteoblasts on the titanium surface used as a control or on anodic oxides formed at low voltages showed many thick stress fibres and intense focal contacts. Alkaline phosphatase (ALP) activity of the cells did not show any correlation with surface characteristics of anodic oxides.     

Characterization of wild-type recombinant Bet v 1a as a candidate vaccine against birch pollen allergy

BACKGROUND: We describe the production in Escherichia coli as a recombinant protein of clinical grade wild-type Bet v 1a (rBet v 1a), to be used as a candidate vaccine against birch pollen allergy. METHODS: This recombinant protein was purified by hydrophobic interaction and ion exchange chromatography and characterized by SDS-PAGE, immunoprint and circular dichroism in parallel with natural Bet v 1 (nBet v 1) purified from a birch pollen extract. We also compared rBet v 1 and nBet v 1 for their capacity to induce histamine release from basophils and to stimulate T lymphocyte proliferation. RESULTS: rBet v 1a appears in SDS-PAGE as an 18-kDa monomeric protein, whereas purified nBet v 1 comprises a mixture of isoforms (resolving as three distinct bands and six spots after 1-dimensional and 2-dimensional electrophoresis, respectively). Both recombinant and natural purified Bet v 1 molecules are recognized by IgE from birch pollen-allergic patients as well as anti-Bet v 1 murine monoclonal antibodies, suggesting that the recombinant protein is correctly folded in a native configuration. Circular dichroism analysis confirmed that the two Bet v 1 molecules exhibit similar 3-dimensional structures, even if rBet v 1a appears more compact and stable in thermodenaturation/renaturation experiments. Both rBet v 1 and nBet v 1 induce the degranulation of sensitized basophils and proliferation of Bet v 1-specific T lymphocytes in a similar manner. CONCLUSIONS: On the basis of these structural and biological properties, rBet v 1a is a valid candidate vaccine against birch pollen allergy, currently evaluated in humans.     

Long-term safety study of levalbuterol administered via metered-dose inhaler in patients with asthma.

BACKGROUND: Previous studies have raised concerns regarding the safety of regular use of beta2-agonists for treating asthma. Few studies have explored the safety of at least 1 year of use of racemic albuterol, and none have examined long-term dosing of levalbuterol. OBJECTIVE: To examine the long-term safety of levalbuterol hydrofluoroalkane (HFA) vs racemic albuterol HFA administered via metered-dose inhaler (MDI) in patients with stable asthma. METHODS: Patients with mild to moderate asthma (mean forced expiratory volume in 1 second [FEVI], 68.3% of predicted) 12 years or older participated in a multicenter, parallel-group, open-label study. Patients were randomized to levalbuterol HFA MDI (90 microg; 2 actuations of 45 microg; n = 496) or racemic albuterol HFA MDI (180 microg; 2 actuations of 90 microg; n = 250) for 52 weeks of 4 times daily dosing. The primary end point was the incidence of postrandomization adverse events. Asthma exacerbations and pulmonary parameters were also assessed. RESULTS: The overall incidence of adverse events was similar for levalbuterol (72.0%) and racemic albuterol (76.8%). Rates of beta-mediated adverse events, serious adverse events, and discontinuations because of adverse events were low (<15%) and were comparable between groups. Rates of asthma adverse events for levalbuterol and racemic albuterol were 18.3% and 19.6%, respectively. Mean percentage of predicted FEV1 improved after dosing and was stable for both groups. CONCLUSION: In this trial, up to 52 weeks of regular use of levalbuterol HFA MDI or racemic albuterol HFA MDI was well tolerated, and no deterioration of lung function was detected during the study period.     

Inharmonicity detection

Detection of mistuned partials in otherwise harmonic complex tones was investigated in naïve subjects of three different age groups. Signals were presented at constant sensation level to compensate for differences in hearing sensitivity and to specifically examine age-related changes in inharmonicity perception. Performance was measured under two conditions, monaural signal presentation and dichotic signal-noise presentation, with the latter aiming at the influence of contralateral distractor sounds. Stimuli were complex tones with ten harmonics and 125-Hz fundamental frequency. Mistuning detection was measured for the first, second, fourth, and eighth harmonic. In a three-interval, three-alternative forced-choice procedure, subjects were required to distinguish a complex tone containing one mistuned partial from two reference tones, with all partials at their harmonic frequencies. Thresholds were measured as the amount of frequency shift necessary for the mistuning to be detected. Performance deteriorated moderately with age for the two higher partials tested, but not for the lower ones. Thresholds for dichotic signal/noise presentation did not differ significantly from monaural ones in any of the age groups. Results are discussed in relation to hypotheses of harmonicity perception in auditory scene analysis and with respect to the investigation of patients suffering form respective deficits due to acquired brain lesions.     

Osteochondral grafts in growing rabbits

Summary In 60-day (group A) and 90-day (group B) old rabbits a standardized osteochondral graft was taken from the distal articular surface of the femur and replanted immediately. Five animals in each group were observed at 9 different times between 3 days and 6 months. On histological and autoradiographic (³⁵S-sulphate) examination the following were found: In group A there was no ³⁵S uptake in the deep layers of the articular cartilage between 3 days and 1 week; in most cases there was normal articular cartilage in the transplants at 2 weeks to 6 months. In group B later changes (3 weeks—6 months), affecting the greater part of the articular cartilage, were observed. These changes appeared to be irreversible and were found in about 1/3 of the cases. The other 2/3 showed completely normal articular cartilage.Fluorescence-microscopic (after tetracycline administration), microradiographic and microangiographic (Indian ink) studies revealed the following: Revascularization of the subchondral bone took place after 3–5 days. The ossification process in the subchondral area was restored within 5–7 days. The osseous part of the transplants healed by primary bone union within 1–2 weeks. The revascularization took place more rapidly in group A.At the longest observation times (8 weeks and 6 months) in both groups slight flattening of the transplant area was seen, probably as a result of slightly retarded growth of the bone within the transplant.Supported by grants from the Swedish Medical Research Council, Project No. 17 X-138-08 A.