[Immune profile of elderly patients admitted in a geriatric short care unit]. / Étude du profil immunitaire de sujets âgés hospitalisés en unité de court séjour gériatrique.

INTRODUCTION: Immunosenescence embraces the whole of age-induced changes observed in the immunomodulatory functions of a living organism, and is mostly characterized by a decrease in cell-mediated immunity and important modifications of the immunological repertoire. The impact of the pathology on ageing immunity is poorly understood and few data are available on the immunological status of old polypathological patients. METHODS: We report the results of a prospective study aiming at characterizing several established immunological parameters in patients of 75 years old or more, and admitted for diverse pathologies in a unit of acute geriatric ward. RESULTS: Among the 51 included patients (35 women and 16 men), 90% displayed poly-pathologies. We found a prevalence of 86% of immunological abnormalities, with lymphopenia among 41% of the patients (<1500/mm(3)) and abnormal lymphocytes phenotypes among 95% of the oldest patients (>85 years). A strong skewing towards memory T lymphocytes (CD45RO+) over naive T lymphocytes (CD45RA+) was found in 80% of the cases and inverted CD4/CD8 T cells ratio was observed in 12% of our patients. Vitamin D insufficiency (<30ng/ml), which is frequent among the patients (94%), is a predictive factor for T and B cell lymphopenia. CONCLUSION: Immunological abnormalities are frequent in this frail population and lymphopenia, in particular, could constitute a reinforcing factor of fragility. Vitamin D deficiency could also affect elderly patients' immunity.

[Immunosenescence and infections, myth or reality?]. / Immunosénescence et infections, mythe ou réalité?

The susceptibility of elderly people to infectious diseases is usually associated to increasing risk factors found in young adults. However, the role of immune function ageing is associated with the decline of immune function but this decline is not homogenous. Some functions such as the cellular immune system are altered but others are enhanced such as innate immunity. The important events of immune ageing are modifications of lymphocyte subsets with accumulation of memory cells, decrease in proliferative response, and a chronic inflammatory state. The chronic antigenic load throughout life is responsible for gaps in the antigenic system with a greater sensitivity to new antigens. These immune system changes are all the more important that diseases are severe and that denutrition is associated. These diseases will speed up the ageing process. The interaction between immunosenescence and pathology is an important phenomenon to consider. This review outlines the immune system changes due to ageing, their relationship with diseases of the aged patient, and the consequences of these modifications on vaccination effectiveness.

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.

Is there a predictable relationship between surface physical-chemical properties and cell behaviour at the interface?

There is much interest in predicting and controlling the outcome of interaction between artificial surfaces and living cells. However, although there is an impressive amount of information on the behaviour of many cell populations deposited on a variety of surfaces, there is presently no available theory to explain or even summarize these data. Indeed, it is not even obvious that such a theory may exist. The aim of the present review is to emphasize the problems encountered when one attempts to build such a theory. Three sequential steps of cell surface interactions are considered: 1) protein adsorption is a preliminary step liable to involve irreversible interaction between the surface and several hundreds of molecular species occurring in blood or plasma. 2) the second step is the formation of adhesive bonds. Several theoretical frameworks were suggested to account for this step, including DLVO theory, physical chemistry of surfaces, and formation of specific ligand-receptor bonds. It is concluded that present evidence supports the latter approach, although this involves serious difficulties. 3) The last step is the triggering of a specific cell program such as apoptosis, proliferation, migration, differentiation or activation. Recent evidence suggests that in addition to the nature and amount of stimulated surface receptors, additional cues such as substratum mechanical or topographical properties may significantly affect cell behaviour.

Cell fitting to adhesive surfaces: A prerequisite to firm attachment and subsequent events.

Cell adhesion usually involves extensive shape reorganization. This process is important because i) it is required for efficient cross-linking of interacting surfaces by adhesion receptors the length of which does not exceed several tens of nanometers and ii) it influences subsequent cell differentiation and activation. This review focuses on the initial phase of cell deformation, preceding the extensive reorganization process known as spreading. This first phase includes local flattening at the micrometer scale and membrane alignment at the nanometer level, resulting in fitting of the cell to an adhesive surface. Three main points are considered. First, experimental methods available to study cell apposition to a surface are described, with an emphasis on interference reflection microscopy. Second, selected experimental evidence is presented to show that there is a quantitative relationship between "adhesiveness" and "contact extension", and some theoretical models aimed at relating these parameters are briefly sketched. Third, experimental data on the kinetics of initial contact extension are described and possible mechanisms for driving this extension are discussed, including nonspecific forces, receptor-mediated interactions, active cell movements or passive membrane fluctuations. It is concluded that both passive physical phenomena and random active cell movements are possible candidates for the initial triggering of contact extension.

Diffusion of microspheres in shear flow near a wall: use to measure binding rates between attached molecules.

The rate and distance-dependence of association between surface-attached molecules may be determined by monitoring the motion of receptor-bearing spheres along ligand-coated surfaces in a flow chamber (Pierres et al., Proc. Natl. Acad. Sci. U.S.A. 95:9256-9261, 1998). Particle arrests reveal bond formation, and the particle-to-surface distance may be estimated from the ratio between the velocity and the wall shear rate. However, several problems are raised. First, data interpretation requires extensive computer simulations. Second, the relevance of standard results from fluid mechanics to micrometer-size particles separated from surfaces by nanometer distances is not fully demonstrated. Third, the wall shear rate must be known with high accuracy. Here we present a simple derivation of an algorithm permitting one to simulate the motion of spheres near a plane in shear flow. We check that theoretical predictions are consistent with the experimental dependence of motion on medium viscosity or particle size, and the requirement for equilibrium particle height distribution to follow Boltzman's law. The determination of the statistical relationship between particle velocity and acceleration allows one to derive the wall shear rate with 1-s(-1) accuracy and the Hamaker constant of interaction between the particle and the wall with a sensitivity better than 10(-21) J. It is demonstrated that the correlation between particle height and mean velocity during a time interval Deltat is maximal when Deltat is about 0.1-0.2 s for a particle of 1.4-microm radius. When the particle-to-surface distance ranges between 10 and 40 nm, the particle height distribution may be obtained with a standard deviation ranging between 8 and 25 nm, provided the average velocity during a 160-ms period of time is determined with 10% accuracy. It is concluded that the flow chamber allows one to detect the formation of individual bonds with a minimal lifetime of 40 ms in presence of a disruptive force of approximately 5 pN and to assess the distance dependence within the tens of nanometer range.

Glycocalyx modulation is a physiological means of regulating cell adhesion.

Here we present experimental evidence that phagocytic cells use modulation of specific components of their glycocalyx to regulate their binding capacity. Particles coated with antibodies specific for the CD32 medium affinity IgG receptor were driven along human monocytic THP-1 cells (expressing CD32) in a flow chamber operated at low shear rate. Surprisingly, only minimal adhesion was observed. However, when cells were activated by exposure to fibronectin-coated surfaces and/or soluble &ggr; interferon, adhesion efficiency was dramatically increased, whereas the apparent glycocalyx thickness displayed 20% decrease, and the surface density of CD43/leukosialin carbohydrate epitopes displayed 30-40% decrease on activated cells. The existence of a causal link between adhesion increase and glycocalyx alteration was strongly supported by the finding that (i) both phenomena displayed similar kinetics, (ii) an inverse relationship between THP-1 cell binding capacity and glycocalyx density was demonstrated at the individual cell level, and (iii) adhesion enhancement could not be ascribed to an increased binding site density or improved functional capacity of activated cells. Additional experiments revealed that cell-to-particle adhesion resulted in delayed (i.e. more than a few minutes) egress of CD43/leukosialin from contact areas. Since the time scale of particle attachment was less than a second, surface mobility should not affect the potential of CD43 to impair the initial step of adhesion. Finally, studies performed with fluorescent lectins suggested that THP-1 cell activation and increased adhesive potential were related to a decrease of O-glysosylation rather than N-glycosylation of surface glycoproteins.

Integrin (alpha) and beta subunit contribution to the kinetic properties of (alpha)2beta1 collagen receptors on human keratinocytes analyzed under hydrodynamic conditions.

The adhesion of keratinocytes to type I collagen or laminin 5 was studied in a laminar flow chamber. These experiments provided an insight into the binding kinetics of integrins in their natural environment and the effects of monoclonal antibodies specific for (alpha) and beta chains. Cells driven by a force too low to alter the natural lifetime of a single bond displayed multiple arrests. Studying the frequency and duration of these arrests yielded fairly direct information on the rate of bond formation (on-rate) and dissociation (off-rate). Off-rate values obtained on collagen or laminin 5 (0.06 seconds-1) were tenfold lower than values determined on selectins. Bond stability was strongly regulated by anti-beta1 chain antibodies since the off-rate was decreased sixfold by activating antibody TS2/16 and increased fivefold by inhibitory antibodies Lia1/2 or P4C10, whereas neutral antibody K20 had no effect on this parameter. Binding frequencies were not significantly changed by all these antibodies. In contrast, both binding frequency and off-rate were altered by antibodies specific for the (alpha)2 chain, suggesting that these antibodies interfered with ligand recognition and also with the ligand-beta1 chain interactions responsible for bond stabilization. The latter hypothesis was supported by the finding that the partial alteration of (alpha)2 chain function by inhibiting antibodies was corrected by anti-beta1 chain antibody TS2/16. These results could not be ascribed to allosteric changes of the functional region of beta1 integrin subunits regulated by TS2/16 since there was no competition between the binding of TS2/16 and anti-(alpha)2 chain antibodies. Interpreted within the framework of current concepts of integrin-ligand binding topology, these data suggest that ligand-alpha chain interactions may be qualitatively important in ligand recognition and also influence the formation of the ligand-beta1 subunit bonding involved in stabilization of the ligand-integrin complex by regulating its dissociation rate.

[Adhesion molecules and cancer]. / Les molécules d'adhésion en cancérologie.

INTRODUCTION: This review was aimed at summarizing recent advances in the understanding of cell adhesion in order to discuss the possible relevance of new knowledge to the exploration of cancer patients and elaboration of therapeutic strategies. CURRENT KNOWLEDGE AND KEY POINTS: During the last 10 years, many adhesion molecules were identified, thus allowing to determine their tissue distribution and functional regulation. The concept of adhesiveness was refined. It is now well known that adhesive rate (i.e., the minimal contact time required for bond formation) and binding strength (i.e., the minimal force required to detach bound cells) are distinct parameters. They may be regulated independently, and influence the cell behavior in different ways. It is now possible to achieve accurate control of tumor cell adhesiveness, either by inhibiting an adhesive mechanism (through monoclonal antibodies, competitive ligands, or inhibition of receptor expression with antisense strategy or gene knock-out) or by promoting a binding mechanism (with receptor transfection or pro-inflammatory stimulation). FUTURE PROSPECTS AND PROJECTS: Recent progress opens new possibilities for diagnosis and treatment. First, the interpretation of experimental data may be improved. Cell adhesive behavior is not entirely accounted for by the density of membrane adhesion receptors. Indeed, adhesion is influenced by receptor connection to the cytoskeleton and structure of the cell coat. An adhesion receptor may be anti-metastatic through an increase in tumor cohesion and cell differentiation, or pro-metastatic, through facilitation of cell migration towards a target tissue. New therapeutic strategies may include anti-adhesive procedure aimed at preventing metastasis formation. The potential importance of a better control of inflammatory processes is also emphasized in view of the influence of these processes on the expression of adhesion molecules.

Studying receptor-mediated cell adhesion at the single molecule level.

Cell adhesion is essentially mediated by specific interactions between membrane receptors and ligands. It is now apparent that the mere knowledge of the on- and off-rate of association of soluble forms of these receptors and ligands is not sufficient to yield accurate prediction of cell adhesive behavior. During the last few years, a variety of complementary techniques relying on the use of hydrodynamic flow, atomic force microscopy, surface forces apparatus or soft vesicles yielded accurate information on i) the dependence of the lifetime of individual bonds on applied forces and ii) the distance dependence of the association rate of bound receptors and ligands. The purpose of this review is, first to recall the physical significance of these parameters, and second to describe newly obtained results. It is emphasized that molecular size and flexibility may be a major determinant of the efficiency of receptor mediated adhesion, and this cannot be studied by conventional methods dealing with soluble molecules.

Experimental study of the interaction range and association rate of surface-attached cadherin 11.

We describe a method allowing quantitative determination of the interaction range and association rate of individual surface-attached molecules. Spherical beads (1.4 micro(m) radius) were coated with recombinant outer domains of the newly described classical type II cadherin 11, a cell adhesion molecule. Beads were driven along cadherin-coated surfaces with a hydrodynamic force of approximately 1 pN, i.e., much less than the mechanical strength of many ligand-receptor bonds. Spheres displayed periods of slow motion interspersed with arrests of various duration. Particle position was monitored with 50 Hz frequency and 0.025 micro(m) accuracy. Nearly 1 million positions were recorded and processed. Comparison between experimental and computer-simulated trajectories suggested that velocity fluctuations might be related quantitatively to Brownian motion perpendicular to the surface. The expected amplitude of this motion was of order of 100 nm. Theoretical analysis of the relationship between sphere acceleration and velocity allowed simultaneous determination of the wall shear rate and van der Waals attraction between spheres and surface. The Hamaker constant was estimated at 2.9 x 10(-23) J. The frequency of bond formation was then determined as a function of sphere velocity. Experimental data were consistent with the view that the rate of association between a pair of adhesion molecules was approximately 1.2 x 10(-3) s-1 and the interaction range was approximately 10 nm. It is concluded that the presented methodology allows sensitive measurement of sphere-to-surface interactions (with approximately 10 fN sensitivity) as well as the effective range and rate of bond formation between individual adhesion molecules.

Use of a laminar flow chamber to study the rate of bond formation and dissociation between surface-bound adhesion molecules: effect of applied force and distance between surfaces.

It has recently been shown that much information on the behaviour of surface-bound adhesion molecules could be obtained by monitoring the motion of receptor-coated particles along ligand-derivatized surfaces in the presence of a hydrodynamic force of a few pN. This procedure is expected to allow direct monitoring of the formation and dissociation of individual bonds. We present experimental results on the interaction between streptavidin-coated spheres (1.4 microns diameter) and control or biotinylated mica surfaces in a laminar flow chamber. Moving spheres are found to display numerous arrests whose frequency is markedly increased (5-13-fold) in the presence of biotin groups. For a given shear rate, the binding frequency is strongly dependent on the sphere-surface separation. Indeed, this frequency displayed a 14-fold decrease when the velocity increased from 7 to 15 microns s-1 for a wall shear rate of 20 s-1. Furthermore, the lifetime of observed arrests was of the order of several seconds, i.e. 5-50-fold higher than previously determined on models such as selectin-ligand, CD2-CD48 or cadherin-cadherin. Finally, this lifetime did not decrease when the wall shear rate was increased from ca. 10 to 40 s-1.

The dependence of the association rate of surface-attached adhesion molecules CD2 and CD48 on separation distance.

The kinetics of bond formation between spherical beads coated with CD48 and CD2-derivatized surfaces was studied with a flow chamber. For a given shear rate, the binding frequency was exquisitively sensitive to the particle velocity. Flow equations were used to derive the particle-to-surface distance from the velocity, thus yielding a relationship between this distance and the binding rate. Numerical values of the binding site densities allowed absolute determination of the rate of association between two individual molecules as a function of the distance between attachment points. In our model, this rate was about 0.03 s-1 at 10 nm separation, and it was inversely proportional to the cube of the distance.

Interest of image processing in cell biology and immunology.

Microscopy is a basic tool for cell biologists. Recent progress of electronics and computer science made powerful methodologies for digital processing of microscopic images easily available. These methods allowed impressive increase of the power of conventional microscopy. Dramatic image enhancement may be achieved by combination of filtering techniques, computer-based deblurring and contrast enhancement. Quantitative treatment of digitized images allows absolute determination of the density of different components of the observed sample, including antigens, intracellular calcium and pH. Morphometric studies are also greatly facilitated by image processing techniques. The capture of fast phenomena may be performed by transfer of small portion of microscopic images into computer memory as well as particular use of confocal microscopy. Finally, improved display of experimental data through coded colors or other procedures may enhance the amount of information that can be conveyed by visual examination of microscopical images. The purpose of the present review is to describe the basic principles of image processing and exemplify the power of this approach with a variety of illustrated applications to conventional, fluorescence or electron microscopy as well as confocal microscopy.

Determination of the lifetime and force dependence of interactions of single bonds between surface-attached CD2 and CD48 adhesion molecules.

We studied single molecular interactions between surface-attached rat CD2, a T-lymphocyte adhesion receptor, and CD48, a CD2 ligand found on antigen-presenting cells. Spherical particles were coated with decreasing densities of CD48-CD4 chimeric molecules then driven along CD2-derivatized glass surfaces under a low hydrodynamic shear rate. Particles exhibited multiple arrests of varying duration. By analyzing the dependence of arrest frequency and duration on the surface density of CD48 sites, it was concluded that (i) arrests were generated by single molecular bonds and (ii) the initial bond dissociation rate was about 7.8 s-1. The force exerted on bonds was increased from about 11 to 22 pN; the detachment rate exhibited a twofold increase. These results agree with and extend studies on the CD2-CD48 interaction by surface plasmon resonance technology, which yielded an affinity constant of approximately 10(4) M-1 and a dissociation rate of > or = 6 s-1. It is concluded that the flow chamber technology can be an useful complement to atomic force microscopy for studying interactions between isolated biomolecules, with a resolution of about 20 ms and sensitivity of a few piconewtons. Further, this technology might be extended to actual cells.

Measuring bonds between surface-associated molecules.

Adhesive interactions play an essential role in immune function. Much information on these phenomena was recently obtained by applying sophisticated methods such as the surface forces apparatus, atomic force microscopy, lipid vesicle-based technology or flow chambers. In the present review it is shown that the use of hydrodynamic flow allows quantitative study of the formation and dissociation of individual molecular bonds between receptor-bearing cells or particles and ligand-derivatized surfaces. In addition, it should be possible to determine particle-surface interaction forces with subpiconewton sensitivity and nanometer resolution. Data analysis shows that the classical concepts of bond strength, or association and dissociation rates must be reexamined in order to achieve a correct understanding of the behavior of individual molecules.

Use of thermal fluctuations to study the length and flexibility of ligand-receptor bonds.

We describe an experimental approach yielding new information on the behavior of ligand-receptor bonds. Spherical particles of 1.4 microns radius were coated with anti-rabbit immunoglobulin monoclonal antibodies and deposited on surfaces derivatized with rabbit immunoglobulins. Brownian motion was studied. When particles where bound through multiple bonds, the mean square displacement during a 1 s interval was 0.0038 micron2 as compared to 0.245 micron2 for unbound particles. Under the same conditions, the mean square displacement of particules coated with limiting dilutions of binding sites and bound by single molecular bonds was 0.0774 micron2. Results are compatible with the concept that the latter particles behaved as spheres transiently bound to the substratum by links of 2.7 nm length, allowing brownian oscillations with an angular amplitude of 0.062 radian.

Measuring the lifetime of bonds made between surface-linked molecules.

It is not well known how the kinetic constants of association between soluble receptors and ligands may be used to predict the behavior of these molecules when they are bound to cell surfaces. Spherical beads were coated with varying densities of anti-rabbit immunoglobulin monoclonal antibodies and driven along glass surfaces derivatized with rabbit anti-dinitrophenol. Particle motion was analyzed. The velocity, attachment frequency, and duration of binding events were determined on individual particles. It was found that i) beads exhibited frequent arrests lasting between a few tenths of a second and more than one minute; ii) when antibodies were diluted, the median arrest duration remained fairly constant (approximately 1 s) whereas binding frequency varied as the first power of the antibody concentration, suggesting that most particle arrests were due to the formation of a single bond; iii) when the shear rate was increased 7-fold, the duration of transient binding events remained constant. The disruptive force exerted on attachment points was estimated to range between about 6 and 37 piconewtons; and iv) the distribution of arrest durations suggested that binding was not a monophasic reaction but involved at least one intermediate step. Therefore, transient binding events reflected the formation of unstable associations that are not detected with standard techniques.

Influence of surface charges on cell adhesion: difference between static and dynamic conditions.

We tested the hypothesis that nonspecific repulsion, as a result of electrostatic forces and (or) steric stabilization effects, impaired adhesion more efficiently under dynamic than under static conditions. Cells from the human monocytic line THP1 were plated on a glass surface. Spherical particles bearing monoclonal antibodies specific for antigens expressed by THP1 cells (CD11b, CD18, CD35, CD64) were then added and adhesion was quantified. The effect of neuraminidase treatment of THP1 cells was also studied. Adhesion was then measured in a flow chamber under low shear flow (wall shear rate was 11 or 22 s-1), allowing a quantitative determination of cell adhesion frequency. The following conclusions were obtained: (i) under static conditions, neuraminidase treatment had little effect on adhesion (only CD18-mediated interaction was significantly increased at 4 degrees C after enzyme treatment); (ii) under dynamic conditions, neuraminidase treatment significantly increased binding; (iii) surprisingly, there was no clear relationship between the length of adhesion molecules involved in the interaction and binding efficiency; and (iv) such parameters as cell shape and topographical distribution of adhesion molecules may strongly influence adhesion under flow. It is concluded that a dynamic reorganization of the pericellular matrix following intercellular contact may play an important role in regulating adhesion.

Dynamic adhesion of CD8-positive cells to antibody-coated surfaces: the initial step is independent of microfilaments and intracellular domains of cell-binding molecules.

Cell adhesion is a multistep, metabolically active process usually requiring several minutes or even hours to complete. This results in the formation of strong bonds that cannot be ruptured by mechanical forces encountered by living cells in their natural environment. However, the first seconds after contact formation are much more sensitive to external conditions and may be the critical step of adhesion. This step is very difficult to monitor without disturbing the observed system. We addressed this problem by studying the interaction between anti-CD8-coated or control surfaces and murine lymphoid cell lines bearing wild-type CD8 molecules, or genetically engineered molecules bearing extracellular CD8 domains and transmembranar and intracytoplasmic domains of class I histocompatibility molecules, or with extensive deletion of intracytoplasmic domains. We used a new method that consisted of monitoring the motion of cells driven along adhesive surfaces by a hydrodynamic force weaker than the reported strength of single ligand-receptor bonds, but sufficient to make free cells move with an easily detectable velocity of several micrometers per second. Cells exhibited short-term (< or = 0.5 s) adhesions to the surface with a frequency of about one event per 30-s period of contact. These events did not require specific antigen-antibody bonds. However, when anti-CD8 were present, strong adhesion was achieved within < 1 s, since most arrests were longer than a standard observation period of 1 min. This bond strengthening was not affected by cytochalasin, and it did not require intact intracellular domains on binding molecules. It is concluded that the initial step in strong adhesion may be viewed as a passive, diffusion-driven formation of a new specific bonds.