Quantitative elastography of the uterine cervix as a predictor of preterm delivery.

OBJECTIVE: To evaluate the correlation between preterm delivery before 37 weeks of gestation and ultrasound elastography strain measurement of cervical stiffness. STUDY DESIGN: In this prospective study, 182 pregnant women were examined vaginally by ultrasound elastography from a mid-sagittal plane. Cervical length was measured and strain was calculated in four regions of interest on the anterior cervical lip. First, the software was validated by intraobserver variability. Second, strain and strain ratios were calculated with adjusted software presets and correlated to the outcome of spontaneous preterm delivery (sPTD). RESULT: A total of 8928 regions of interest (ROIs) and 6696 ratios were evaluated. The median gestational age at examination was 26 ± 6.1 weeks. A median maternal age of 33 ± 5.6 and a medial parity of 1 ± 0.9 were observed. Intra-Class-Correlation values in validation phase ranged from 0.893 to 0.967. The prevalence of sPTD was 11.9%. Strain ratio Rselective was identified as the best predictor of preterm delivery. Rselective values >0.89 were associated with preterm delivery with a sensitivity of 0.59 and a specificity of 0.86 (odds ratio=1.474 for an increase of 0.1 in Rselective; P=0.002). CONCLUSION: Ultrasound elastography strain measurement of cervical stiffness is correlated with the predictability of preterm delivery.

An inhaled tumor necrosis factor-alpha-derived TIP peptide improves the pulmonary function in experimental lung injury.

INTRODUCTION: The lectin-like domain of TNF-α enhances the fluid clearance across the alveolar barrier. For experimental purposes, the lectin-like domain can be mimicked by a synthetic peptide representing the TIP-motif of TNF-α. The present study aims to assess the acute effect of TIP on the pulmonary function in a porcine model of acute respiratory distress syndrome (ARDS). METHODS: Lung injury was induced in 16 pigs (25-27 kg) by bronchoalveolar lavage followed by injurious ventilation. Following randomisation, either nebulised TIP (1 mg/kg; AP301, APEPTICO, Vienna, Austria) or water for injection (control group) was administered. During 5 h of monitoring, the extravascular lung water index (EVLWI), the quotient of partial pressure of oxygen and inspired oxygen concentration (PaO(2) /FiO(2) ) and the pulmonary shunt fraction were repetitively assessed. The data were evaluated by an analysis of variance including Bonferroni-Holm correction. RESULTS: Comparable baseline conditions in both groups were achieved. Ventilatory parameters were standardised in both groups. In the TIP group, a significant reduction of the EVLWI and a simultaneous increase in the PaO(2) /FiO(2) ratio was shown (each P < 0.0001). No changes in the control group were observed (EVLWI: P = 0.43, PaO(2) /FiO(2) : P = 0.60). The intergroup comparison demonstrates a significant advantage of TIP inhalation over placebo (EVLWI: P < 0.0001, PaO(2) /FiO(2) : P = 0.004, shunt fraction: P = 0.0005). CONCLUSIONS: The inhalation of TIP induces an amelioration of clinical surrogate parameters of the lung function in a porcine lung injury model. By mimicking the lectin-like domain, the synthetic TIP peptide AP301 is an innovative approach as supportive therapy in ARDS.

In vitro analysis of birch-pollen-associated food allergy by use of recombinant allergens in the basophil activation test.

BACKGROUND: Basophil activation is associated with the expression of CD63. In birch-pollen-associated food allergy to celery, carrot and apple, Bet v 1, Api g 1, Dau c 1 and Mal d 1 are major allergens. Recombinant allergens have not yet been used in the CD63-based basophil activation test (BAT). OBJECTIVE: To evaluate the feasibility of using recombinant allergens in the BAT in the diagnosis of allergy to apple, carrot and celery and to compare results with routine tests, i.e. skin prick tests (SPTs) and specific IgE. METHODS: Thirty-two patients with an oral allergy syndrome induced by apple, carrot or celery and 22 controls were studied. SPTs were performed with native foods. Specific IgE was determined by the CAP method and basophil activation by flowcytometry upon double staining with anti-IgE/anti-CD63 monoclonal antibodies after incubating with purified recombinant Bet v 1, Bet v 2, Api g 1, Dau c 1 and Mal d 1. RESULTS: By the combined use of the BAT and the CAP method, sensitization to Bet v 1 and Bet v 2 was detected in 100 and 25% of all subjects, respectively. Sensitivity of specific IgE for apple, carrot and celery was 60, 70 and 75% with corresponding specificities of 64, 86 and 82%. Sensitivity of the BAT for Mal d 1, Dau c 1 and Api g 1 was 75, 65 and 75% with corresponding specificities of 68, 100 and 77%. CONCLUSIONS: The BAT using recombinant allergens provides a valuable new in vitro method for the detection of sensitization to foods. Although double-blind placebo-controlled food challenges remain the gold standard to confirm food allergy, the CD63-based BAT with recombinant allergens may supplement routine tests for allergy diagnosis.

The basophil activation test in wasp venom allergy: sensitivity, specificity and monitoring specific immunotherapy.

BACKGROUND: As in vitro diagnosis of wasp venom sensitization by specific serum IgE has a sensitivity of only 60-80%, additional in vitro tests are desirable. Basophil activation is associated with the expression of CD63 and its measurement has been proposed as a novel in vitro test for immediate-type allergy. Furthermore, to date, no in vitro test exists to monitor successful specific immunotherapy (SIT) with wasp venom. Therefore, the potentially harmful sting challenge is still recommended. OBJECTIVE: We compared the CD63-based basophil activation test (BAT) in the diagnosis of wasp venom allergy with skin tests and measurement of specific IgE. Furthermore, we investigated whether BAT can predict the outcome of the sting challenge in patients on SIT. METHODS: Fifty patients with a systemic reaction caused by a wasp sting and 20 controls were studied. Intracutaneous tests were performed with wasp and bee venom in the suspected allergics. Specific IgE was determined by the CAP-FEIA method and basophil activation by flow cytometry upon double staining with anti-IgE/anti-CD63 mAb. Twenty-five patients were sting challenged 6 months after starting SIT and the BAT was repeated before challenge. RESULTS: Sensitivity of the intracutaneous tests, specific IgE and BAT was 100, 76, and 92%, respectively. Specificity of specific IgE and the BAT was 85 and 80%, respectively. The cut-off for a positive BAT was 15% CD63+ basophils. There was a positive correlation between IgE reactivity to wasp venom and the number of CD63+ basophils (r = 0.65). Although no patient had a systemic reaction upon sting challenge, in most subjects basophil activation did not decrease when compared with the BAT before SIT. CONCLUSIONS: Quantitation of basophil activation by CD63 expression is a valuable new in vitro method for diagnosis of allergy to hymenopteran venoms. The CD63-based BAT is a helpful tool for the complementation of routine diagnostic tests such as specific IgE as it increases sensitivity of in vitro detection of sensitization. However, this in vitro method does not offer an alternative to the sting challenge in monitoring successful SIT.

Differential management of Ca2+ oscillations by anterior pituitary cells: a comparative overview.

Most electrical and ionic properties of anterior pituitary cells are common to all pituitary cell types; only gonadotropes exhibit a few cell specific features. Under basal conditions, the majority of pituitary cells in vitro, irrespective of their cell type, display spontaneous action potentials and [Ca2+]i transients that result from rhythmic Ca2+ entry through L-type Ca2+ channels. The main function of these action potentials is to maintain cells in a readily activable responsive state. We propose to call this state a 'pacemaker mode', since it persists in the absence of extrinsic stimulation. When challenged by hypothalamic releasing hormones, cells exhibit two distinct response patterns: amplification of pacemaker activity or shift to internal Ca2+ release mode. In the internal Ca2+ release mode, [Ca2+]i oscillations are not initiated by entry of external Ca2+, but by release of Ca2+ from intracellular stores. In somatotropes and corticotropes, GHRH or CRH triggers the pacemaker mode in silent cells and amplifies it in spontaneously active cells. In contrast, in gonadotropes GnRH activates the internal Ca2+ release mode in silent cells and switches already active cells from the pacemaker to the internal Ca2+ release mode. Interestingly, homologous normal and tumoral cells display the same type of activity in vitro, in the absence or presence of hypothalamic hormones. Pacemaker and internal Ca2+ release modes are likely to serve different purposes. Pacemaker activity allows long-lasting sequences of [Ca2+]i oscillations (and thus sustained periods of secretion) that stop under the influence of hypothalamic inhibitory peptides. In contrast, the time during which cells can maintain internal Ca2+ release mode depends upon the importance of intracellular Ca2+ stores. This mode is thus more adapted to trigger secretory peaks of large amplitude and short duration. On the basis of these observations, theoretical models of pituitary cell activity can be proposed.

Endogenous pacemaker activity of rat tumour somatotrophs.

1. Cells derived from a rat pituitary tumour (GC cell line) that continuously release growth hormone behave as endogenous pacemakers. In simultaneous patch clamp recordings and cytosolic Ca2+ concentration ([Ca2+]i) imaging, they displayed rhythmic action potentials (44.7 +/- 2.7 mV, 178 +/- 40 ms, 0.30 +/- 0.04 Hz) and concomitant [Ca2+]i transients (374 +/- 57 nM, 1.0 +/- 0.2 s, 0.27 +/- 0.03 Hz). 2. Action potentials and [Ca2+]i transients were reversibly blocked by removal of external Ca2+, addition of nifedipine (1 microM) or Ni2+ (40 microM), but were insensitive to TTX (1 microM). An L-type Ca2+ current activated at -33.6 +/- 0.4 mV (holding potential (Vh), -40 mV), peaked at -1.8 +/- 1.3 mV, was reduced by nifedipine and enhanced by S-(+)-SDZ 202 791. A T/R-type Ca2+ current activated at -41.7 +/- 2.7 mV (Vh, -80 or -60 mV), peaked at -9.2 +/- 3.0 mV, was reduced by low concentrations of Ni2+ (40 microM) or Cd2+ (10 microM) and was toxin resistant. Parallel experiments revealed the expression of the class E calcium channel alpha1-subunit mRNA. 3. The K+ channel blockers TEA (25 mM) and charybdotoxin (10-100 nM) enhanced spike amplitude and/or duration. Apamin (100 nM) also strongly reduced the after-spike hyperpolarization. The outward K+ tail current evoked by a depolarizing step that mimicked an action potential reversed at -69. 8 +/- 0.3 mV, presented two components, lasted 2-3 s and was totally blocked by Cd2+ (400 microM). 4. The slow pacemaker depolarization (3.5 +/- 0.4 s) that separated consecutive spikes corresponded to a 2- to 3-fold increase in membrane resistance, was strongly Na+ sensitive but TTX insensitive. 5. Computer simulations showed that pacemaker activity can be reproduced by a minimum of six currents: an L-type Ca2+ current underlies the rising phase of action potentials that are repolarized by a delayed rectifier and Ca2+-activated K+ currents. In between spikes, the decay of Ca2+-activated K+ currents and a persistent inward cationic current depolarize the membrane, activate the T/R-type Ca2+ current and initiate a new cycle.

Growth hormone-releasing hormone triggers pacemaker activity and persistent Ca2+ oscillations in rat somatotrophs.

1. The effects of brief applications of growth hormone-releasing hormone (GHRH) to male rat somatotrophs in culture were analysed with the perforated patch clamp technique to record changes in potential or with fura-2 imaging techniques to measure variations of cytosolic Ca2+ concentration ([Ca2+]i). 2. Silent somatotrophs (n = 61) had a mean resting potential of -37 +/- 1 mV and a mean basal [Ca2+]i of 30 +/- 4 nM. Brief GHRH applications (30 nM, 40 s) triggered rhythmic action potentials (23.6 +/- 0.9 mV, 613 +/- 82 ms, 0.21 +/- 0.02 Hz) and [Ca2+]i increase (to 352 +/- 30 nM) followed by rhythmic [Ca2+]i transients (to 138 +/- 6 nM) that persisted up to 90 min after the last GHRH application. Both action potentials and [Ca2+]i transients were totally and reversibly blocked by removing external Ca2+ or Na+ or by adding inorganic Ca2+ channel blockers or nifedipine (3 microM). 3. Somatostatin (1-300 nM), carbamylcholine (0.1-1 microM) and muscarine (0.1-1 microM) each had a dose-dependent inhibitory effect, from a decrease of Ca2+ spike duration and frequency to a complete block of the GHRH-evoked action potentials. 4. The present results show that somatotrophs in culture have intrinsic membrane properties that allow them to sustain a pacemaker activity and subsequent long-lasting sequences of [Ca2+]i oscillations triggered by short pulses of GHRH and inhibited by somatostatin and muscarinic agonists.

The hypoglycemic sulphonylurea tolbutamide increases N-methyl-D-aspartate- but not kainate-activated currents in hippocampal neurons in culture.

The effects of the hypoglycemic sulphonylurea tolbutamide, a marker of K(+)-ATP channels, on the N-methyl-D-aspartate- (NMDA) and kainate-activated currents were studied in rat hippocampal neurons in culture, using the patch-clamp technique in a whole-cell configuration. Tolbutamide (500 microM) reversibly increased the peak amplitude and the steady state level of NMDA- but not kainate-evoked currents. This effect was not glycine dependent as it was observed at low and saturated concentrations of glycine. The affinity of the NMDA receptor-channel complex for glycine did not change in the presence of tolbutamide. The action of tolbutamide on the NMDA-activated current was not mediated by K(+)-ATP channels since CsCl was added intracellularly at concentrations which completely blocked all K+ channels. Possible mechanisms explaining the effect of tolbutamide via the modulation of intracellular messengers are discussed.