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.     

Slow event-related brain activity of aphasic patients and controls in word comprehension and rhyming tasks

Slow event-related potentials (ERP) were examined in healthy and aphasic subjects in two-stimulus designs comprising a word comprehension and a rhyming task. Aphasics, though selected to perform above chance level, made significantly more errors and responded more slowly than controls, although canonical correlations did not indicate a statistical relationship between performance measures and ERP amplitudes. A discriminant analysis of ERP amplitudes distinguished the groups for the slow wave (SW; 0.5-1.0 s post-S1 onset) in the word comprehension, for the SW and the initial contingent negative variation (iCNV; 1.0-2.0 s post-S1 onset) in the rhyming task. Similarly for both tasks, ERP topography showed left-anterior predominance of the negative SW and iCNV in controls, whereas participants with aphasia showed smaller anterior and larger left-posterior amplitudes. The centroparietal terminal CNV (tCNV; 1 s pre-S2) was smaller in participants with aphasia than in controls, but similar in topography. Results suggest left-anterior activation for those language processes that were presumably provoked in the present tasks, like lexical access, or phonological encoding. The pattern of participants with aphasia may indicate effects of language impairment and recovery, but also consequences of the brain damage.     

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.     

Cyclic and non-cyclic variations of spinal cord temperature related with temperature regulation in pigeons

1. The temperature of the spinal cord (Tvc) was measured in unanaesthetized pigeons at different ambient temperatures (Ta). 2. In short-term experiments spontaneous or noncyclic variations of Tvc at constant (20 and 10 degrees C) and changing Ta were correlated with the amplitude of the electromyogram EMG) which indicates shivering, i.e., heat production. At constant Ta no unequivocal correlation between Tvc (and also Tskin) and EMG was found. During ambient cooling there was often an increase of Tvc which resulted in a positive correlation whereas there was a negative correlation to Tskin. 3. In long-term experiments (24h, LD 12:12Y cyclic variations of Tvc were measured at different Ta and ocrrelated with O2-consumption, i.e., heat production. As with body temperature Tvc was lowered during the dark phase of the diurnal cycle. In the light phase both Tvc and heat production increased with decreasing Ta which results in a positive correlation. In the dark phase there was a smaller increase in heat production but a decrease in Tvc, i.e., a negative correlation. 4. The results show that natural variations of Tvc are often positively ocrrelated with heat production. This is in contrast to experimental changes of Tvc where a clear negative correlation to heat production can be observed.     

The subchondral bone plate of the femoral head in adult rabbits

Summary The mineralized structures and spontaneous remodelling of the subcondral bone-plate in the femoral head of adult rabbits of different ages were studied by microradiography and fluorescence microscopy after administration of Tetracycline between 8 months and 2 days before death. In animals over 1 year of age the subchondral bone-plate was constructed of evenly mineralized osteones, located mainly in the immediate vicinity of the calcified articular cartilage and lamellar bone bordering on the medullary cavity. In some small areas lamellar bone was the only bone tissue separating the medullary cavity from the calcified articular cartilage. In adult animals between 8 and 10 months of age the subchondral boneplate was absent in minor areas, chiefly in the lateral parts of the femoral head. In the fluorescence microscopic study in animals older than 1 year (a) the osteones in the subchondral bone showed a very low labelling frequency, (b) fluorescence was observed mainly on the lamellar bone surfaces bordering on the medullary cavity and (c) the labelling in the cortical part of the subchondral bone was still present up to 8 months after administration of the Tetracycline. The cancellous bone of the epiphysis showed a low labelling frequency which was of the same order as that in the lamellar surfaces belonging morphologically to the subchondral bone plate.From these observations it was concluded that (1) the cortical part of the subchondral bone-plate in rabbits over 1 year of age undergoes a low degree of spontaneous remodelling and (2) the cancellous bone of the epiphysis and the lamellar bone belonging morphologically to the subchondral bone-plate undergo low but similarly spontaneous remodelling. Duplicate fluorescence of the tidemark observed in a number of animals was considered to indicate that the mineralization of the articular cartilage with increasing age slowly progresses towards the articular surface.     

Interaction of B7RP-1 with ICOS Negatively Regulates Antigen Presentation by B Cells

Stimulation of T cells through the T cell receptor is insufficient for optimal T cell activation. A second activation signal is necessary, being usually provided by the costimulatory molecule CD28. Recently, additional costimulatory pathways have been identified, including inducible costimulator (ICOS) and its ligand B7RP-1. We have examined the role of the B7RP-1/ICOS costimulatory pathway on antigen presentation by B cells, using the I-A^k and I-E^k-positive CH27 B cell line and several different T cell lines. We found that CH27 expressed B7RP-1 and PD-L1 whereas the T cell lines expressed ICOS and PD-1. In the presence of HEL, the T cell hybridomas C10 and 3A9 released IL-2, which is indicative of antigen-specific T cell activation by the CH27 cells. Unexpectedly, blocking antibodies for B7RP-1 and ICOS enhanced the IL-2 response in both T cells. As expected, an increase in the production of IL-2 was seen when blocking antibodies for PD-1 were used. Blocking with antibodies for I-A^k, CD28, B7.1, and B7.2 lead to a decrease in IL-2 production. Additionally we tested a Th1 and a Th2 T cell clone. Blockade of B7RP-1/ICOS lead to an increased IFN- response in Th1 cells (A.E7) and an increased IL-4 response in Th2 cells (D10.G4.1). Intracellular staining also showed an increase in cytokine production when the B7RP-1/ICOS pathway was blocked. In conclusion, the B7RP-1/ICOS pathway is negatively regulating T cell activation by B cells and may play a role similar to that of the PD-L1/PD-1 pathway.