Early contacts between T lymphocytes and activating surfaces.

Cells continually probe their environment to adapt their behaviour. A current challenge is to determine how they analyse nearby surfaces and how they process information to take decisions. We addressed this problem by monitoring human T lymphocyte attachment to surfaces coated with activating anti-CD3 or control anti-HLA antibodies. Interference reflection microscopy allowed us to monitor cell-to-surface apposition with a few nanometre vertical resolution during the first minutes following contact. We found that (i) when a cell fell on a surface, contact extension was preceded by a lag of several tens of seconds. (ii) During this lag, vertical membrane undulations seemed to generate transient contacts with underlying surfaces. (iii) After the lag period, the contact area started increasing linearly with a rate of about 1.5 µm(2) s(-1) on activating surfaces and about 0.2 µm(2) s(-1) on control surfaces. (iv) Concomitantly with lateral surface extension, the apparent distance between cell membranes and surfaces steadily decreased. These results are consistent with the hypothesis that the cell decision to spread rapidly on activating surfaces resulted from the integration of information yielded by transient contacts with these surfaces generated by membrane undulations during a period of about 1 min.

Cationic ruthenium allenylidene complexes as catalysts for ring closing olefin metathesis

A series of well accessible cationic ruthenium allenylidene complexes of the general type [(eta6-arene)(R3P)RuCl(=C=CR'2)]+ X- is described which constitute a new class of pre-catalysts for ring closing olefin metathesis reactions (RCM) and provide an unprecedented example for the involvement of metal allenylidenes in catalysis. They effect the cyclization of various functionalized dienes and enynes with good to excellent yields and show a great tolerance towards an array of functional groups. Systematic variations of their basic structural motif have provided insights into the essential parameters responsible for catalytic activity which can be enhanced further by addition of Lewis or Bronsted acids, by irradiation with UV light, or by the adequate choice of the "non-coordinating" counterion X-. The latter turned out to play a particularly important role in determining the rate and selectivity of the reaction. A similarly pronounced influence is exerted by remote substituents on the allenylidene residue which indicates that this ligand (or a ligand derived thereof) may remain attached to the metal throughout the catalytic process. X-ray crystal structures of the catalytically active allenylidene complexes 3b.PF6 and 15.OTf as well as of the chelate complex 10 required for the preparation of the latter catalyst are reported.

Airway occlusion pressure to titrate positive end-expiratory pressure in patients with dynamic hyperinflation.

BACKGROUND: Although the use of external positive end-expiratory pressure (PEEP) is recommended for patients with intrinsic PEEP, no simple method exists for bedside titration. We hypothesized that the occlusion pressure, measured from airway pressure during the phase of ventilator triggering (P0.1t), could help to indicate the effects of PEEP on the work of breathing (WOB). METHODS: Twenty patients under assisted ventilation with chronic obstructive pulmonary disease were studied with 0, 5, and 10 cm H2O of PEEP while ventilated with a fixed level of pressure support. RESULTS: PEEP 5 significantly reduced intrinsic PEEP (mean +/- SD, 5.2 +/- 2.4 cm H2O at PEEP 0 to 3.6 +/- 1.9 at PEEP 5; P < 0.001), WOB per min (12. 6 +/- 6.7 J/min to 9.1 +/- 5.9 J/min; P = 0.003), WOB per liter (1.2 +/- 0.4 J/l to 0.8 +/- 0.4 J/l; P < 0.001), pressure time product of the diaphragm (216 +/- 86 cm H2O. s-1. min-1 to 155 +/- 179 cm H2O. s-1. min-1; P = 0.001) and P0.1t (3.3 +/- 1.5 cm H2O to 2.3 +/- 1.4 cm H2O; P = 0.002). At PEEP 10, no further significant reduction in muscle effort nor in P0.1t (2.5 +/- 2.1 cm H2O) occurred, and transpulmonary pressure indicated an increase in end-expiratory lung volume. Significant correlations were found between WOB per min and P0.1t at the three levels of PEEP (P < 0.001), and between the changes in P0.1t versus the changes in WOB per min (P < 0.005), indicating that P0.1t and WOB changed in the same direction. A decrease in P0.1 with PEEP indicated a decrease in intrinsic PEEP with a specificity of 71% and a sensitivity of 88% and a decrease in WOB with a specificity of 86% and a sensitivity of 91%. CONCLUSION: These results show that P0.1t may help to assess the effects of PEEP in patients with intrinsic PEEP.

Clinical evaluation of a computer-controlled pressure support mode.

We have designed a computerized system providing closed-loop control of the level of pressure support ventilation (PSV). The system sets itself at the lowest level of PSV that maintains respiratory rate (RR), tidal volume (VT), and end-tidal CO(2) pressure (PET(CO(2))) within predetermined ranges defining acceptable ventilation (i.e., 12 < RR < 28 cycles/min, VT > 300 ml [> 250 if weight < 55 kg], and PET(CO(2)) < 55 mm Hg [< 65 mm Hg if chronic CO(2) retention]). Ten patients received computer-controlled (automatic) PSV and physician-controlled (standard) PSV, in random order, during 24 h for each mode. An estimation of occlusion pressure (P(0.1)) was recorded continuously. The average time spent with acceptable ventilation as previously defined was 66 +/- 24% of the total ventilation time with standard PSV versus 93 +/- 8% with automatic PSV (p < 0.05), whereas the level of PSV was similar during the two periods (17 +/- 4 cm H(2)O versus 19 +/- 6 cm H(2)O). The time spent with an estimated P(0.1) above 4 cm H(2)O was 34 +/- 35% of the standard PSV time versus only 11 +/- 17% of the automatic PSV time (p < 0.01). Automatic PSV increased the time spent within desired ventilation parameter ranges and apparently reduced periods of excessive workload.

Changes in the work of breathing induced by tracheotomy in ventilator-dependent patients.

Tracheotomy is widely performed on ventilator-dependent patients, but its effects on respiratory mechanics have not been studied. We measured the work of breathing (WOB) in eight patients before and after tracheotomy during breathing at three identical levels of pressure support (PS): baseline level (PS-B), PS + 5 cm H2O (PS+5), and PS - 5 cm H2O (PS-5). After the procedure, we also compared the resistive work induced by the patients' endotracheal tubes (ETTs) and by a new tracheotomy cannula in an in vitro bench study. A significant reduction in the WOB was observed after tracheotomy for PS-B (from 0.9 +/- 0.4 to 0.4 +/- 0.2 J/L, p < 0.05), and for PS-5 (1.4 +/- 0.6 to 0.6 +/- 0.3 J/L, p < 0.05), with a near-significant reduction for PS+5 (0.5 +/- 0.5 to 0.2 +/- 0.1 J/L, p = 0.05). A significant reduction was also observed in the pressure-time index of the respiratory muscles (181 +/- 92 to 80 +/- 56 cm H2O. s/min for PS-B, p < 0.05). Resistive and elastic work computed from transpulmonary pressure measurements decreased significantly at PS-B and PS-5. A significant reduction in occlusion pressure and intrinsic positive end-expiratory pressure (PEEP) was also observed for all conditions, with no significant change in breathing pattern. Three patients had ineffective breathing efforts before tracheotomy, and all had improved synchrony with the ventilator after the procedure. In vitro measurements made with ETTs removed from the patients, with new ETTs, and with the tracheotomy cannula showed that the cannula reduced the resistive work induced by the artificial airway. Part of these results was explained by a slight, subtle reduction of the inner diameter of used ETTs. We conclude that tracheotomy can substantially reduce the mechanical workload of ventilator-dependent patients.

Gravity effects on upper airway area and lung volumes during parabolic flight.

We measured upper airway caliber and lung volumes in six normal subjects in the sitting and supine positions during 20-s periods in normogravity, hypergravity [1.8 + head-to-foot acceleration (Gz)], and microgravity ( approximately 0 Gz) induced by parabolic flights. Airway caliber and lung volumes were inferred by the acoustic reflection method and inductance plethysmography, respectively. In subjects in the sitting position, an increase in gravity from 0 to 1. 8 +Gz was associated with increases in the calibers of the retrobasitongue and palatopharyngeal regions (+20 and +30%, respectively) and with a concomitant 0.5-liter increase in end-expiratory lung volume (functional residual capacity, FRC). In subjects in the supine position, no changes in the areas of these regions were observed, despite significant decreases in FRC from microgravity to normogravity (-0.6 liter) and from microgravity to hypergravity (-0.5 liter). Laryngeal narrowing also occurred in both positions (about -15%) when gravity increased from 0 to 1.8 +Gz. We concluded that variation in lung volume is insufficient to explain all upper airway caliber variation but that direct gravity effects on tissues surrounding the upper airway should be taken into account.

NéoGanesh: a working system for the automated control of assisted ventilation in ICUs.

Automating the control of therapy administered to a patient requires systems which integrate the knowledge of experienced physicians. This paper describes NéoGanesh, a knowledge-based system which controls, in closed-loop, the mechanical assistance provided to patients hospitalized in intensive care units. We report on how new advances in knowledge representation techniques have been used to model medical expertise. The clinical evaluation shows that such a system relieves the medical staff of routine tasks, improves patient care, and efficiently supports medical decisions regarding weaning. To be able to work in closed-loop and to be tested in real medical situations, NéoGanesh deals with a voluntarily limited problem. However, embedded in a powerful distributed environment, it is intended to support future extensions and refinements and to support reuse of knowledge bases.

Evaluation of a knowledge-based system providing ventilatory management and decision for extubation.

We evaluated whether a knowledge-based system (KBS) connected to a ventilator in pressure support mode could correctly predict the ability of patients to tolerate total withdrawal from ventilatory support. The KBS was designed to continuously adapt ventilatory assistance to the needs of the patient, to manage a strategy of gradually decreasing ventilatory assistance, and to indicate when the patient was able to breathe without assistance. Thirty-eight patients for whom weaning was being considered were evaluated using a conventional battery of parameters, including weaning criteria, tolerance of a T-piece trial, and outcome 48h after permanent withdrawal of ventilation. The results of this evaluation were compared with the suggestions made by the KBS at the end of a period of KBS-driven mechanical ventilation inserted in the conventional weaning procedure. The positive predictive value of the KBS was 89%, versus 77% for the conventional procedure and 81% for the rapid shallow breathing index alone. The KBS correctly predicted the course of five patients who tolerated a T-piece trial but required ventilation within 48 h. We conclude that our KBS ensured appropriate patient management during the weaning period and improved our ability to predict responses to weaning.

Evaluation of carbon dioxide rebreathing during pressure support ventilation with airway management system (BiPAP) devices.

The purpose of this study was to evaluate whether carbon dioxide (CO2) rebreathing occurs in acute respiratory failure patients ventilated using the standard airway management system (BiPAP pressure support ventilator; Respironics; Murrysville, Pa) with positive inspiratory airway pressure and a minimal level of positive end-expiratory pressure (PEEP) and whether any CO2 rebreathing may be efficiently prevented by the addition of a nonrebreathing valve to the BiPAP system circuit. In the first part of the study, the standard device was tested on a lung model with a nonrebreathing valve (BiPAP-NRV) and with the usual Whisper Swivel connector (BiPAP-uc). With the BiPAP-uc device, the resident volume of expired air in the inspiratory circuit at the end of expiration (RVEA) was 55% of the tidal volume (VT) when the inspiratory pressure was 10 cm H2O and the frequency was at 15 cycles per minute. The BiPAP-NRV device efficiently prevented CO2 rebreathing but resulted in a slight decrease in VT, which was due to a significant increase in external PEEP (2.4 vs 1.3 cm H2O) caused by the additional expiratory valve resistance. For similar reasons, both the pressure swing necessary to trigger pressure support and the imposed expiratory work were increased in the lung model when the nonrebreathing valve was used. In the second part of the study, seven patients weaned from mechanical ventilation were investigated using a randomized crossover design to compare three situations: pressure support ventilation with a conventional intensive care ventilator (CIPS), BiPAP system use, and BiPAP-NRV. When we compared the BiPAP system use with the other two systems, we observed no significant effect on blood gases but found significant increases in VT, minute ventilation, and work of breathing. These findings are experimental and are clinical evidence that significant CO2 rebreathing occurs with the standard BiPAP system. This drawback can be overcome by using a non-rebreathing valve, but only at the expense of greater expiratory resistance.

Effect of chronic administration of cyclosporin A on hepatic uptake and biliary secretion of bromosulfophthalein in rat.

Cyclosporin A (CyA) decreases bile flow and bile salt secretion in the rat. The purpose of this study was to examine the influence of CyA on the hepatic transport of bromosulfophthalein (BSP). Male Sprague-Dawley rats were injected with CyA at the daily dose of 10 mg/kg (treated animals) or solvent (controls) during three weeks. Hepatic uptake of BSP (assessed by the plasma disappearance curve of the dye) and biliary secretion during infusions (95.5 and 178 nmol/min/100 g) were examined in both groups. Administration of CyA resulted in a decrease in both bile flow and BSP biliary secretion at the two infusion rates used. BSP plasma disappearance rate was significantly lower in treated animals than in controls. Conjugation of the dye was unaffected by CyA. There was no modification in ALT activity or in liver histology. These data show that chronic administration of CyA in rats decreases both hepatic uptake and biliary secretion of BSP. Thus, the inhibitory effect of CyA on biliary secretion is not limited to bile salts but also is observed with other cholephilic substances.

Deficient induction of sulfobromophthalein conjugating activity by phenobarbital in hamster liver.

Administration of phenobarbital, a known inducer of glutathione S-transferase activity in rat liver, failed to stimulate sulfobromophthalein (BSP) conjugation by liver cytosol in hamsters. The latter displayed poor ability to conjugate this substrate, despite very high glutathione-conjugating activity with the broad-spectrum substrate 1-chloro-2,4-dinitrobenzene (CDNB). Of the six substrates tested, in this species, 1,2-epoxy-3-(4-nitrophenoxy)propane (ENPP) was the only one whose conjugation was greatly enhanced by phenobarbital (+172%). Nevertheless, hamsters proved as responsive to phenobarbital induction as rats, since it increased their relative liver weight and microsomal enzyme activity. The deficient induction of liver BSP-conjugating activity observed with phenobarbital is consistent with the finding that it did not affect the hepatic transport of this substrate in hamsters.

Inhibition of liver glutathione S-transferase activity in rats by hypolipidemic drugs related or unrelated to clofibrate.

The effects of in vivo administration of six hypolipidemic drugs on rat liver glutathione S-transferase activity were compared. This activity was measured with sulfobromophthalein (BSP), 1,2-dichloro-4-nitrobenzene (DCNB) or 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. Except for the nicotinic acid derivative ethanolamine oxiniacate, all the compounds tested significantly reduced it, whether or not they were related to clofibrate. The hepatic glutathione concentration either remained unchanged or only increased slightly after treatment with the various drugs. When measured, the maximal excretion rate of bile BSP dropped significantly, but not that of phenol-3,6-dibromophthalein (DBSP). Hepatic dye uptake and storage were not impaired. These results show that hypolipidemic drugs of the peroxisome proliferator type inhibit rat liver glutathione S-transferase activity and may reduce transport of anions conjugated with glutathione before excretion.

Impairment of hepatic glutathione S-transferase activity as a cause of reduced biliary sulfobromophthalein excretion in clofibrate-treated rats.

Administration of clofibrate reduced the maximal excretion rate of bile sulfobromophthalein (BSP) in rats but left that of phenol-3,6-dibromophthalein (DBSP) unchanged. This decrease in liver transport of BSP was due to reduced bile excretion of conjugated BSP. Hepatic uptake and storage of this dye were not impaired. Liver glutathione S-transferase activity in vitro, measured with BSP, 1,2-dichloro-4-nitrobenzene (DCNB) or 1-chloro-2, 4-dinitrobenzene (CDNB) was significantly reduced. This alteration in liver conjugating activity was probably not related to a modification of the hepatic GSH pool, since the GSH level was unchanged or only increased slightly after clofibrate treatment. Detection of this inhibition required at least two daily doses of clofibrate. Inhibition was dose-related and lasted for several days after cessation of the drug. In clofibrate-treated rats, Lineweaver-Burk plots showed a reduced Vmax for both the BSP and GSH substrates. These results suggest that clofibrate decreases hepatobiliary transport of BSP by lowering glutathione S-transferase activity in the liver.