Detection of alterations in all three exons of the peripherin/RDS gene in Swedish patients with retinitis pigmentosa using an efficient DGGE system.

AIMS: To develop a sensitive mutation screening procedure suitable for routine analysis of the peripherin/RDS gene, and to estimate the nature and prevalence of peripherin/RDS gene mutations in Swedish patients with autosomal dominant retinitis pigmentosa. METHODS: To make the method as sensitive as possible, as many as eight segments, covering the three exons and the flanking intron sequences of the peripherin/RDS gene, were analysed by denaturing gradient gel electrophoresis. A group of 38 Swedish patients with a clinical diagnosis of autosomal dominant retinitis pigmentosa were screened for mutations in the peripherin/RDS gene. RESULTS: Three point mutations were found in four of the patients and five polymorphisms were defined. One mutation in exon 1, R172W, has been described previously in other ethnic groups as causing a macular degeneration. Another mutation, in exon 2 and causing the substitution F211L, was found in two unrelated patients. A third mutation, resulting in the likely non-pathogenic substitution S289L, as well as a polymorphism not reported previously, was found in exon 3. CONCLUSIONS: The screening procedure described allows detection of mutations in all of the exons, including the polymorphic 5' and 3' ends of the gene, and is therefore suitable for routine screening of peripherin/RDS gene defects in patients with autosomal dominant retinitis pigmentosa. The frequency of mutations found in the Swedish patient group indicates that defects in the peripherin/RDS gene might be a more common cause of autosomal dominant retinitis pigmentosa than was thought previously.     

Cardiac changes during sleep in sleep-deprived infants

OBJECTIVE: To evaluate the influence of a brief period of sleep deprivation on cardiac autonomic controls during sleep in healthy infants. DESIGN: Twelve healthy infants with a median age of 8 weeks (range, 7 to 18 weeks) were recorded polygraphically during a morning and an afternoon nap in a sleep laboratory. They were sleep deprived for approximately 2 hours, either in the morning or in the afternoon, before being allowed to fall asleep. Six infants were sleep deprived before the morning nap, and 6 before the afternoon nap. During both naps, their sleep, breathing, and heart-rate characteristics were continuously recorded. Spectral analysis of heart rate was evaluated as a function of sleep stages. Two major peaks were recognizable: a low-frequency component related to sympathetic and parasympathetic activities and a high-frequency component reflecting parasympathetic tonus. The ratio of low-frequency to high-frequency powers was calculated as an index of sympathovagal interaction. RESULTS: When sleep deprived, the infants had an increase in basal heart rate during non-rapid eye movement sleep (P=.021). With sleep deprivation, the ratio of low-frequency to high-frequency powers increased in non-rapid eye movement sleep (P=.005). These findings were consistent with an increase in sympathetic tone. CONCLUSION: Infants exposed to short-term sleep deprivation manifest changes in cardiac autonomic controls during sleep. These findings could be relevant to mechanisms associated with the sudden infant death syndrome.     

An open,randomized single-centre study to compare the efficacy and convenience of follitropin beta administered by a pen device with follitropin alpha administered by a conventional syringe in women undergoing ovarian stimulation for IVF/ICSI

BACKGROUND: A pen device, similar to an insulin pen, has been recently marketed for the administration of follitropin beta in cartridges. A randomized controlled trial was performed to compare the efficacy and convenience of this pen device delivering follitropin beta with a conventional syringe delivering follitropin alpha. METHODS: A total of 200 patients needing IVF/ICSI treatment and willing to self-inject were enrolled in the study. All subjects had ovarian stimulation according to a long protocol and were randomized to the pen or the conventional syringe group during down-regulation by means of a computer-generated randomization list using random numbers. Patients were asked to fill in a daily local tolerance book after each injection. On the day of hCG the patients scored a Visual Analogue Scale (VAS) for pain and convenience. RESULTS: The average duration, total dose of recombinant FSH and number of cumulus oocyte complexes retrieved were 10.8/12.0 days (P = 0.001), 1880/2226 IU (P < 0.001) and 15.2/13.1 respectively in the pen device and conventional syringe groups; the presence of pain after the daily injection was significantly higher in the conventional syringe group (P = 0.027); the visual analogue scale score was similar for pain but significantly more convenient for the pen device (P < 0.001). The live birth rate per embryo transfer was 32.9 and 34.4% respectively in the pen device and conventional syringe groups. CONCLUSIONS: Self-injection with the pen device is safe and easy, more convenient and less painful for the patient, requires less FSH and shortens the treatment duration.     

Galectin-1 is overexpressed in nasal polyps under budesonide and inhibits eosinophil migration

Because of the importance of galectins for various cellular activities, the influence of the glucocorticoid budesonide on the level of expression of galectins-1 and -3 was investigated in human nasal polyposis. Ten nasal polyps obtained from surgical resection were maintained for 24 hours in the presence of various concentrations of budesonide. As quantitatively demonstrated by means of computer-assisted microscopy, 250 ng/ml (the highest dose tested) induced a pronounced increase of galectin-1 expression. This feature was observed in nasal polyps from allergic patients but not in those from nonallergic patients. Since eosinophils represent the main inflammatory cell population in nasal polyps, we investigated the effect of galectin-1 on their migration levels by means of quantitative phase-contrast computer-assisted videomicroscopy. Our results show that galectin-1 (coated on plastic supports) markedly reduced the migration levels of eosinophils in comparison to P-selectin. On the cellular level, marked modifications in the polymerization/depolymerization dynamics of the actin cytoskeleton (as revealed by means of computer-assisted fluorescence microscopy) and, to a much lesser extent, an increase in the adhesiveness of eosinophils to tested substrata were detectable. The present study therefore reveals a new galectin-1-mediated mechanism of action for glucocorticoid-mediated anti-inflammatory effects.     

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.     

Interinstitutional variations of sensitometric curves of radiographic dosimetric films

Depth and field size dependence of the sensitometric curves of radiographic films have been studied by various groups. Limited information is, however, available on the magnitude of the variations in sensitometric curves applied in clinical practice in different institutions. In this study we assessed in a systematic way the effect of the various parameters influencing the shape of the sensitometric curve: batch composition, irradiation conditions, film processing, and film scanning. Two types of film, Kodak X-Omat V and CEA TVS, were irradiated, processed, and analyzed in three different institutions. The interinstitutional variation of the sensitometric curves, expressed as the OD variation at 50 cGy, can be up to 32% and is mainly caused by differences in film processing and to a lesser degree to differences in batch composition, film scanning, and irradiation conditions. For the Kodak films, the average OD difference at 50 cGy between the three institutions is 17% as a result of differences in batch composition and 25% due to differences in processing conditions. For the CEA films these data are 6% and 24%, respectively. The long-term variation of the sensitometric curves of KODAK films in one institution was smaller than the differences in batch composition between the three institutions. The sensitometric curves of CEA films showed in one institution a large variation with time; the shape gradually varied from sigmoidal to quasilinear. By using relative OD values rather than absolute OD values, variations in sensitometric curves of KODAK films can be reduced to 2%. Consequently, one sensitometric curve is sufficient to derive relative dose values. If processing conditions are well controlled, it might therefore be advantageous to determine the absolute OD only at one or two dose values, in combination with a "universal" relative sensitometric curve.     

Ara h 8, a Bet v 1-homologous allergen from peanut, is a major allergen in patients with combined birch pollen and peanut allergy

BACKGROUND: We recently described patients with soybean allergy mainly mediated by cross-reactivity to birch pollen allergens. A majority of those patients were reported to have peanut allergy. OBJECTIVE: We sought to study the occurrence of peanut allergy in patients allergic to birch pollen and characterized the Bet v 1-homologous peanut allergen Ara h 8. METHODS: Recombinant Ara h 8 was cloned with degenerated primers and expressed in Escherichia coli. Nine Swiss and 11 Dutch patients with peanut and birch pollen allergy and a positive double-blind, placebo-controlled food challenge result to peanut were investigated for IgE reactivity to birch pollen and purified peanut allergens and cross-reactivity between birch and peanut. Ara h 8 stability against digestion and roasting was assessed by means of RAST inhibition. The IgE cross-linking potency of Ara h 8 was tested on the basis of basophil histamine release. RESULTS: During double-blind, placebo-controlled food challenge, all patients experienced symptoms in the oral cavity, progressing to more severe symptoms in 40% of patients. CAP-FEIA detected recombinant (r) Ara h 8-specific IgE in 85%. IgE binding to Ara h 8 was inhibited by Bet v 1 in peanut extract immunoblotting and in RAST inhibition. In EAST inhibition recombinant rAra h 8 inhibited IgE binding to peanut in 4 of 7 tested patient sera. Antipeanut response was dominated by Ara h 8 in 12 of 17 tested patients. Furthermore, our results demonstrate a low stability of Ara h 8 to roasting and no stability to gastric digestion. Basophil histamine release with rAra h 8 was more than 20% in 5 of 7 tested sera. CONCLUSIONS: Peanut allergy might be mediated in a subgroup of our patients by means of cross-reaction of Bet v 1 with the homologous peanut allergen Ara h 8.