Storage of blood for in vitro generation of dendritic cells.

BACKGROUND: Since the development of techniques to cultivate DC from peripheral blood, there has been a great deal of interest in the use of these cells in immunotherapeutic strategies. In a clinical setting, delays often occur between when blood is drawn and when it is processed. We therefore investigated the effect of overnight storage on the yield, morphology and phenotype of DC cultured from the peripheral blood of healthy volunteers. METHOD: Blood was processed either immediately, or after storage for 24 h in the fridge (4 degrees C) or at room temperature (RT, 20 degrees C). Samples were compared for starting cell number, DC yield and characteristics (morphology and phenotype). RESULTS: The number of PBMC that could be obtained was significantly lower from the refrigerated samples compared with both the freshly processed sample and that stored at RT. Samples processed after overnight storage at RT yielded cells morphologically identical to DC cultured from freshly processed samples. Only when samples were both stored and processed cold did the cultured cells not have typical DC morphology. DC cultured from the refrigerated samples showed a significant reduction in MHC II expression compared with samples processed fresh or stored at RT. This expression increased slightly when the sample was first warmed. Total DC yield and the percentage yield of cultured DC was not significantly different for any of the groups. DISCUSSION: We conclude that, if immediate processing of blood for in vitro generation of DC is not possible, samples should be stored at room temperature (approximately 20 degrees C).

Evaluation of the stability of (99m)Tc-ECD and stabilized (99m)Tc-HMPAO stored in syringes.

OBJECTIVE: To determine and compare the stability of (99m)Tc-ECD and stabilized (99m)Tc-HMPAO when stored in syringes over an 8-h period. METHODS: (99m)Tc-ECD and stabilized (99m)Tc-HMPAO were prepared according to the manufacturers' protocols, with the following exception: eluate less than 60 min old was used to prepare (99m)Tc-HMPAO rather than the recommended 30 min. Once prepared, 185 MBq (5 mCi) of both products were drawn into 5-mL syringes and allowed to sit at room temperature. At 2, 4, 6, and 8 h after preparation, the radiochemical purity (RCP) of the contents of the syringes was determined and compared to the RCP of the products in vials. Retention of activity of each product in syringes was also evaluated by measuring activity remaining in each syringe (and filter, in the case of (99m)Tc-HMPAO) after expressing its contents. RESULTS: The RCP of stabilized (99m)Tc-HMPAO stored in syringes decreased from a mean of 87.7% at 2 h to 74.0% at 8 h after preparation. In contrast, (99m)Tc-ECD retained an RCP of greater than 94% throughout the time tested. The impurity that appeared to increase over time with (99m)Tc-HMPAO was found to be sodium pertechnetate. Total retention of activity remaining in the syringe and filter ranged from 11.6% at 2 h to 9.5% at 8 h for (99m)Tc-HMPAO; the syringe itself retained less than 5% of the total activity at all time periods. (99m)Tc-ECD exhibited 6.2% to 11.3% retention of activity in the syringe. The sorption of sodium pertechnetate to the syringe for the same time period was less than 1%. CONCLUSIONS: (99m)Tc-ECD is a more stable product than stabilized (99m)Tc-HMPAO in a syringe. Both products demonstrate retention of radioactivity in the syringe. Some of this retention may denote sorption of the products to plastic.

A selective N-type calcium channel antagonist protects against neuronal loss after global cerebral ischemia.

Calcium influx is believed to play a critical role in the cascade of biochemical events leading to neuronal cell death in a variety of pathological settings, including cerebral ischemia. The synthetic omega-conotoxin peptide SNX-111, which selectively blocks depolarization-induced calcium fluxes through neuronal N-type voltage-sensitive calcium channels, protected the pyramidal neurons in the CA1 subfield of the hippocampus from damage caused by transient forebrain ischemia in the rat model of four-vessel occlusion. SNX-111 provided neuroprotection when a single bolus injection was administered intravenously up to 24 hr after the ischemic insult. These results suggest that the window of opportunity for therapeutic intervention after cerebral ischemia may be much longer than previously thought and point to the potential use of omega-conopeptides and their derivatives in the prevention or reduction of neuronal damage resulting from ischemic episodes due to cardiac arrest, head trauma, or stroke. Microdialysis studies showed that SNX-111 was 3 orders of magnitude less potent in blocking potassium-induced glutamate release in the hippocampus than the conopeptide SNX-230, which, in contrast to SNX-111, failed to show any efficacy in the four-vessel occlusion model of ischemia. These results imply that the ability of a conopeptide to block excitatory amino acid release does not correlate with its neuroprotective efficacy.

Effect of route of breathing on the ventilatory and arousal responses to hypercapnia in awake and sleeping dogs.

1. The influence of the upper airway on the ventilatory and arousal responses to hypercapnia in wakefulness and sleep was investigated using a chronic animal model. 2. Experiments were performed in five unrestrained dogs trained to sleep naturally in the laboratory. The animal rebreathed through a chronic tracheostoma (thus excluding the upper airway from the breathing circuit), or through the snout (intact upper airway). Resistance to breathing and volume of dead space during quiet tracheal breathing were matched to those in quiet nasal breathing during wakefulness and sleep. CO2 rebreathing tests were performed during wakefulness, rapid eye movement (REM) and non-REM (NREM) sleep, during nasal and tracheal breathing. 3. The ventilatory response to hypercapnia was significantly lower in nasal breathing compared with tracheal breathing, in all behavioural states. This was due to a smaller tidal volume and lower breathing frequency. 4. The ventilatory response to CO2 was lowest during REM sleep, irrespective of route used for breathing. 5. Alveolar partial pressure of CO2 (PA,CO2) level at arousal was identical in NREM nasal and tracheal rebreathing tests. Differences in PA,CO2 levels at arousal between NREM and REM sleep were not significant in nasal tests and only marginally different during tracheal breathing. 6. We conclude that nasal breathing influences the hypercapnic ventilatory response in wakefulness and sleep, and that the presence of CO2 in the upper airway does not affect arousal in NREM and REM sleep.

Restructuring of sleep and reversal of REM-induced supraspinal hypotonia of respiratory muscles following bilateral phrenicotomy.

The long-term effect of diaphragm paralysis on respiratory system function is still not clear. We monitored changes in breathing pattern and the sleep/wake cycle in a dog before and after bilateral phrenicotomy. The post-operative observation extended over 6 months. It was noted that minute ventilation increased during wakefulness and non-REM sleep in the initial 4-6 weeks (compared to pre-surgery period), but decreased during REM sleep, mainly due to inhibition of chest wall and abdominal muscles. These episodes resulted in hypoxemia and frequent arousals. Following this period, there was a restructuring of REM sleep, increasing the frequency of REM sleep and reducing the duration of each REM sleep episode. In addition, the enhanced activity of parasternal and abdominal muscles was persistently seen during REM sleep. These changes in breathing and sleep provided stable ventilation during sleep. We conclude that bilateral phrenicotomy restructures breathing and alters sleep/wake cycle to prevent nocturnal hypoxemia. The mechanisms underlying these changes may reflect plasticity in the control of breathing and REM sleep.