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Immune cells have the ubiquitous capability to migrate disregarding the adhesion properties of the environment, which requires a versatile adaptation of their adhesiveness mediated by integrins, a family of specialized adhesion proteins. Each subtype of integrins has several ligands and several affinity states controlled by internal and external stimuli. However, probing cell adhesion properties on live cells without perturbing cell motility is highly challenging, especially in vivo . Here, we developed a novel in vitro method using micron-size beads pulled by flow to functionally probe the local surface adhesiveness of live and motile cells. This method allowed a functional mapping of the adhesiveness mediated by VLA-4 and LFA-1 integrins on the trailing and leading edges of live human T lymphocytes. We show that cell polarization processes enhance integrin-mediated adhesiveness toward cell rear for VLA-4 and cell front for LFA-1. Furthermore, an inhibiting crosstalk of LFA-1 toward VLA-4 and an activating crosstalk of VLA-4 toward LFA-1 were found to modulate cell adhesiveness with a long-distance effect across the cell. These combined signaling processes directly support the bistable model that explains the emergence of the versatile guidance of lymphocyte under flow. Molecularly, Sharpin, an LFA-1 inhibitor in lymphocyte uropod, was found involved in the LFA-1 deadhesion of lymphocytes; however, both Sharpin and Myosin inhibition had a rather modest impact on adhesiveness. Quantitative 3D immunostaining identified high-affinity LFA-1 and VLA-4 densities at around 50 and 100 molecules/μm 2 in basal adherent zones, respectively. Interestingly, a latent adhesiveness of dorsal zones was not grasped by immunostaining but assessed by direct functional assays with beads. The combination of live functional assays, molecular imaging, and genome editing is instrumental to characterizing the spatiotemporal regulation of integrin-mediated adhesiveness at molecular and cell scales, which opens a new perspective to decipher sophisticated phenotypes of motility and guidance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Robert, Biarnes-Pelicot, Garcia-Seyda, Hatoum, Touchard, Brustlein, Nicolas, Malissen, Valignat and Theodoly.)

Contact with Leishmania leads to a decreases in mononuclear phagocyte adherence to connective tissue. In this work, we studied the early stages of bond formation between VLA4 and fibronectin, measured the kinetics of membrane alignment and the monocyte cytoplasm spreading area over a fibronectin-coated surface, and studied the expression of high affinity integrin epitope in uninfected and Leishmania-infected human monocytes. Our results show that the initial VLA4-mediated interaction of Leishmania-infected monocyte with a fibronectin-coated surface is preserved, however, the later stage, leukocyte spreading over the substrate is abrogated in Leishmania-infected cells. The median of spreading area was 72 [55-89] μm(2) for uninfected and 41 [34-51] μm(2) for Leishmania-infected monocyte. This cytoplasm spread was inhibited using an anti-VLA4 blocking antibody. After the initial contact with the fibronectrin-coated surface, uninfected monocyte quickly spread the cytoplasm at a 15 μm(2) s(-1) ratio whilst Leishmania-infected monocytes only made small contacts at a 5.5 μm(2) s(-1) ratio. The expression of high affinity epitope by VLA4 (from 39 ± 21% to 14 ± 3%); and LFA1 (from 37 ± 32% to 18 ± 16%) molecules was reduced in Leishmania-infected monocytes. These changes in phagocyte function may be important for parasite dissemination and distribution of lesions in leishmaniasis.

Blood leukocytes have a remarkable capacity to bind to and stop on specific blood vessel areas. Many studies have disclosed a key role of integrin structural changes following the interaction of rolling leukocytes with surface-bound chemoattractants. However, the functional significance of structural data and mechanisms of cell arrest are incompletely understood. Recent experiments revealed the unexpected complexity of several key steps of cell-surface interaction: (i) ligand-receptor binding requires a minimum amount of time to proceed and this is influenced by forces. (ii) Also, molecular interactions at interfaces are not fully accounted for by the interaction properties of soluble molecules. (iii) Cell arrest depends on nanoscale topography and mechanical properties of the cell membrane, and these properties are highly dynamic. Here, we summarize these results and we discuss their relevance to recent functional studies of integrin-receptor association in cells from a patient with type III leukocyte adhesion deficiency. It is concluded that an accurate understanding of all physical events listed in this review is needed to unravel the precise role of the multiple molecules and biochemical pathway involved in arrest triggering.

Cell membranes are studded with protrusions that were thoroughly analyzed with electron microscopy. However, the nanometer-scale three-dimensional motions generated by cell membranes to fit the topography of foreign surfaces and initiate adhesion remain poorly understood. Here, we describe the dynamics of surface deformations displayed by monocytic cells bumping against fibronectin-coated surfaces. We observed membrane undulations with typically 5 nm amplitude and 5-10 s lifetime. Cell membranes behaved as independent units of micrometer size. Cells detected the presence of foreign surfaces at 50 nm separation, resulting in time-dependent amplification of membrane undulations. Molecular contact then ensued with apparent cell-membrane separation of 30-40 nm, and this distance steadily decreased during the following tens of seconds. Contact maturation was associated with in-plane egress of bulky molecules and robust membrane fluctuations. Thus, membrane undulations may be the major determinant of cell sensitivity to substrate topography, outcome of interaction, and initial kinetics of contact extension.

Cells continuously probe their environment with membrane receptors, achieving subsecond adaptation of their behaviour [Diez, G., Gerisch, G., Anderson, K., Müller-Taubenberger, A. and Bretschneider, T. (2006) Subsecond reorganization of the actin network in cell motility and chemotaxis. Proc. Natl. Acad. Sci. USA 102, 7601-7606, Shamri, R., Grabovsky, V., Gauguet, J.M., Feigelson, S., Manevich, E., Kolanus, W., Robinson, M.K., Staunton, D.E., von Andrian, U.H. and Alon, R. (2005) Lymphocyte arrest requires instantaneous induction of an extended LFA-1 conformation mediated by endothelium-bound chemokines. Nat. Immunol. 6, 497-606, Jiang, G., Huang, A.H., Cai, Y., Tanase, M. and Sheetz, M.P. (2006) Rigidity sensing at the leading edge through alpha(V)beta(3) integrins and RPTPalpha. Biophys. J. 90, 1804-2006]. Recently, several receptors, including cadherins, were found to bind ligands with a lifetime of order of one second. Here we show at the single molecule level that homotypic C-cadherin association involves transient intermediates lasting less than a few tens of milliseconds. Further, these intermediates transitionned towards more stable states with a kinetic rate displaying exponential decrease with piconewton forces. These features enable cells to detect ligands or measure surrounding mechanical behaviour within a fraction of a second, much more rapidly than was previously thought.

Cell adhesion requires nanometer scale membrane alignment to allow contact between adhesion receptors. Little quantitative information is presently available on this important biological process. Here we present an interference reflection microscopic study of the initial interaction between monocytic THP-1 cells and adhesive surfaces, with concomitant determination of cell deformability, using micropipette aspiration, and adhesiveness, using a laminar flow assay. We report that 1), during the first few minutes after contact, cells form irregular-shaped interaction zones reaching approximately 100 micro m(2) with a margin extension velocity of 0.01-0.02 micro m/s. This happens before the overall cell deformations usually defined as spreading. 2), These interference reflection microscopic-detected zones represent bona fide adhesion inasmuch as cells are not released by hydrodynamic forces. 3), Alignment is markedly decreased but not abolished by microfilament blockade with cytochalasin or even cell fixation with paraformaldehyde. 4), In contrast, exposing cells to hypotonic medium increased the rate of contact extension. 5), Contacts formed in presence of cytochalasin, after paraformaldehyde fixation or in hypotonic medium, were much more regular-shaped than controls and their extension matched cell deformability. 6), None of the aforementioned treatments altered adhesiveness to the surface. It is concluded that adhesive forces and passive membrane deformations are sufficient to generate initial cell alignment to adhesive surfaces, and this process is accelerated by spontaneous cytoskeletally-driven membrane motion.

A laminar flow chamber was used to study single molecule interactions between biotinylated surfaces and streptavidin-coated spheres subjected to a hydrodynamic drag lower than a piconewton. Spheres were tracked with 20 ms and 40 nm resolution. They displayed multiple arrests lasting between a few tens of milliseconds and several minutes or more. Analysis of about 500,000 positions revealed that streptavidin-biotin interaction was multiphasic: transient bound states displayed a rupture frequency of 5.3 s(-1) and a rate of transition toward a more stable configuration of 1.3 s(-1). These parameters did not display any significant change when the force exerted on bonds varied between 3.5 and 11 pN. However, the apparent rate of streptavidin-biotin association exhibited about 10-fold decrease when the wall shear rate was increased from 7 to 22 s(-1), which supports the existence of an energy barrier opposing the formation of the transient binding state. It is concluded that a laminar flow chamber can yield new and useful information on the formation of molecular bonds, and especially on the structure of the external part of the energy landscape of ligand-receptor complexes.

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.

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.

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.

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.

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.