Nonlinear viscoelasticity of concentrated solutions of aggrecan aggregate.

Aggrecan, the major cartilage proteoglycan, is the macromolecular species primarily involved in the resiliency of cartilage tissue. Most aggrecan molecules occur in cartilage extracellular matrix as aggregates. Each aggregate has a supramolecular structure, with many highly anionic, brushlike aggrecan subunits noncovalently bound to a hyaluronan chain. To better examine the viscoelastic properties of aggrecan aggregate, contaminating subunits were removed by exclusion chromatography. At physiologic ionic strength, concentrated solutions of purified aggrecan aggregate exhibit predominantly elastic behavior at small shear strains. However, above a critical strain, gamma c, the shear moduli show a pronounced strain-softening transition, where the storage modulus decreases suddenly, and the loss modulus exhibits a maximum. At small stresses, the creep function is also highly elastic, exhibiting an equilibrium compliance and large recoverable compliance. When the stress is amplified, a discrete transition to viscous flow occurs at a yield stress sigma y. These nonlinear responses are similar to previous observations for close-packed colloidal suspensions of soft spheres, such as microgel or emulsion particles, for which a yield transition occurs when the stress and deformation are sufficient to move a particle past its neighbors.

Droplet growth by coalescence in binary fluid mixtures.

The evolution of the drop-size distribution in immiscible fluid mixtures following well-specified shear histories is investigated by in situ microscopy, allowing determination of the shear-induced coalescence efficiency epsilon. At small capillary number Ca, epsilon is constant, whereas at larger values of Ca, epsilon decreases, in agreement with theory accounting for slight deformation of the drops in close approach. Coalescence causes the drop-size distribution to broaden in general, but greater deformation of the larger drops at high shear rates causes the drop-size distribution to remain narrow.

Rae1 and H60 ligands of the NKG2D receptor stimulate tumour immunity.

Natural killer (NK) cells attack many tumour cell lines, and are thought to have a critical role in anti-tumour immunity; however, the interaction between NK cells and tumour targets is poorly understood. The stimulatory lectin-like NKG2D receptor is expressed by NK cells, activated CD8+ T cells and by activated macrophages in mice. Several distinct cell-surface ligands that are related to class I major histocompatibility complex molecules have been identified, some of which are expressed at high levels by tumour cells but not by normal cells in adults. However, no direct evidence links the expression of these 'induced self' ligands with tumour cell rejection. Here we demonstrate that ectopic expression of the murine NKG2D ligands Rae1beta or H60 in several tumour cell lines results in potent rejection of the tumour cells by syngeneic mice. Rejection is mediated by NK cells and/or CD8+ T cells. The ligand-expressing tumour cells induce potent priming of cytotoxic T cells and sensitization of NK cells in vivo. Mice that are exposed to live or irradiated tumour cells expressing Rae1 or H60 are specifically immune to subsequent challenge with tumour cells that lack NKG2D ligands, suggesting application of the ligands in the design of tumour vaccines.

Expression of natural killer receptor alleles at different Ly49 loci occurs independently and is regulated by major histocompatibility complex class I molecules.

Ly49 receptor genes are expressed by subsets of natural killer (NK) cells in an overlapping fashion, accounting for the capacity of NK subsets to attack host cells that have selectively downregulated self-major histocompatibility complex (MHC) class I molecules. It was shown previously that most NK cells express only one or the other allele of a given Ly49 gene, while a smaller population expresses both alleles. However, the methods used to detect monoallelic and biallelic cells were nonquantitative. Here, new allele-specific antibodies were used to provide the first quantitative examination of biallelic and monoallelic expression of Ly49A and Ly49G2. The results demonstrate conclusively that most Ly49A(+) and Ly49G2(+) NK cells express the corresponding gene in a monoallelic fashion, with a smaller subset expressing both alleles. Unexpectedly, biallelic Ly49A(+) NK cells were more numerous than predicted by completely independent allelic expression, suggesting some heterogeneity among NK progenitors in the potential to express a given Ly49 gene. The data also show that cells expressing one allele of Ly49G2 may express Ly49A from the same or opposite chromosome with equal likelihood, indicating that the expressed allele is chosen independently for different Ly49 genes. Finally, the data demonstrate that biallelic expression of Ly49A or Ly49G2 occurs least frequently in mice that express ligands for these receptors (H-2(d) mice), and most frequently in class I-deficient mice. Thus, biallelic expression of Ly49 genes is regulated by interactions of NK cell progenitors with MHC class I molecules.

Ligands for the murine NKG2D receptor: expression by tumor cells and activation of NK cells and macrophages.

Natural killer (NK) cells attack tumor and infected cells, but the receptors and ligands that stimulate them are poorly understood. Here we report the expression cloning of two murine ligands for the lectin-like receptor NKG2D. The two ligands, H-60 and Rae1 beta, are distant relatives of major histocompatibility complex class I molecules. NKG2D ligands are not expressed by most normal cells but are up-regulated on numerous tumor cells. We show that mouse NKG2D is expressed by NK cells, activated CD8+ T cells and activated macrophages. Expression of either NKG2D ligand by target cells triggers NK cell cytotoxicity and interferon-gamma secretion by NK cells, as well as nitric oxide release and tumor necrosis factor alpha transcription by macrophages. Thus, through their interaction with NKG2D, H-60 and Rae1 beta are newly identified potent stimulators of innate immunity.

Recognition of the class Ib molecule Qa-1(b) by putative activating receptors CD94/NKG2C and CD94/NKG2E on mouse natural killer cells.

The heterodimeric CD94/NKG2A receptor, expressed by mouse natural killer (NK) cells, transduces inhibitory signals upon recognition of its ligand, Qa-1(b), a nonclassical major histocompatibility complex class Ib molecule. Here we clone and express two additional receptors, CD94/NKG2C and CD94/NKG2E, which we show also bind to Qa-1(b). Within their extracellular carbohydrate recognition domains, NKG2C and NKG2E share extensive homology with NKG2A (93-95% amino acid similarity); however, NKG2C/E receptors differ from NKG2A in their cytoplasmic domains (only 33% similarity) and contain features that suggest that CD94/NKG2C and CD94/NKG2E may be activating receptors. We employ a novel blocking anti-NKG2 monoclonal antibody to provide the first direct evidence that CD94/NKG2 molecules are the only Qa-1(b) receptors on NK cells. Molecular analysis reveals that NKG2C and NKG2E messages are extensively alternatively spliced and approximately 20-fold less abundant than NKG2A message in NK cells. The organization of the mouse Cd94/Nkg2 gene cluster, presented here, shows striking similarity with that of the human, arguing that the entire CD94/NKG2 receptor system is relatively primitive in origin. Analysis of synonymous substitution frequencies suggests that within a species, NKG2 genes may maintain similarities with each other by concerted evolution, possibly involving gene conversion-like events. These findings have implications for understanding NK cells and also raise new possibilities for the role of Qa-1 in immune responses.

A new monoclonal antibody reactive with several Ly49 NK cell receptors mediates redirected lysis of target cells.

We produced a novel hamster monoclonal antibody (MAb), 14B11, that recognizes the majority of mouse natural-killer (NK) cells. Transfection studies demonstrated that 14B11 MAb binds a subset of Ly49 receptors, including three putative inhibitory receptors, Ly49F, I, and C. No binding to Ly49A, B, D, or G was detected. In addition, 14B11 was shown to bind the putative activating receptor Ly49H, which required co-transfection of the signaling molecule DAP12 for detectable cell surface expression. Thus, 14B11 is the first reported MAb to bind Ly49H and F. At the functional level, 14B11 MAb enhanced the lysis by IL-2 activated NK cells of an FcR+ target cell line (Daudi), but not an FcR- target cell (EL-4). Because F(ab')2 fragments of 14B11 failed to enhance lytic activity, the enhancement of lysis by intact antibody is apparently due to "redirected lysis," in which stimulatory receptors on the NK cell are bridged by antibody to Fc receptors on the target cell. Cell separation experiments demonstrated that the 14B11-dependent redirected lysis was markedly increased using NK cell populations that had been depleted of Ly49F,+ I,+ or C+ NK cells. Because such depletions are expected to enrich for Ly49H+ NK cells, these results suggest that the enhancement of lysis mediated by 14B11 MAb may be due to stimulation of the activating Ly49H receptor. In conjunction with other anti-Ly49 MAbs, the 14B11 MAb will be useful in further studies of Ly49 receptor function and specificity.

Effects of lipid on the structure and rheology of gels formed by canine submaxillary mucin.

Rheological experiments have shown that canine submaxillary mucin (CSM) forms gels in aqueous solution at low ionic strength and in 6M GdnHCl. Examination of specimens of intact CSM and also its subunits prepared by reduction and carboxymethylation showed that the presence of lipid increases the gel-forming capability, probably as a result of enhancement of the intermolecular hydrophobic interactions. The rheological evidence for gelation is that substantially larger values of the oscillatory storage modulus, G' (omega), and the dynamic complex viscosity, eta*(omega), are observed for lipid-containing CSM. This is backed up by electron micrographs of freeze fractured specimens, where we observe a network morphology in which the cross-links are formed as a result of non-bonded interactions between a number of CSM chains. The intermolecular interactions responsible for gelation probably involve hydrophobic association between the interdigitated oligosaccharides, and/or between the non-glycosylated regions of the protein core, and can occur even in a highly chaotropic medium (6M GdnHCl). In contrast to previous experiments with porcine submaxillary mucin and human tracheobronchial mucin, which form microphase-separated gels in aqueous solution, CSM solutions undergo macroscopic phase separation into polymerrich (gel) and polymer-poor (sol) phases. These data point to stronger hydrophobic interactions in lipid-containing CSM.

Gelation of fractionated canine submaxillary mucin in a chaotropic solvent.

Rheological measurements have been performed on three molecular weight fractions of purified canine submaxillary mucin (CSM) dissolved in the chaotropic solvent 6 M guanidine hydrochloride (GdnHCI). Solutions of the lower molecular weight fractions are viscoelastic sols, and their dynamic moduli can be scaled with respect to molecular weight and concentration according to linear viscoelasticity theory. In contrast, preparations of the highest molecular weight fraction form viscoelastic gels that exhibit an equilibrium shear modulus, Ge', which scales with mucin concentration as Ge' approximately c3. Amino acid and carbohydrate analyses of all three fractions are similar; thus, the differences in rheological behavior are attributed to molecular weight differences, which affect the degree of coil overlap in solutions of a given concentration. These observations demonstrate conclusively that mucin glycoproteins of high molecular weight form gels under conditions in which the mucin chains physically interpenetrate, even when non-covalent intermolecular interactions are extensively disrupted. A comparison of these results with previous studies of purified submaxillary and tracheobronchial mucins indicates that the carbohydrate side-chain length, in addition to molecular weight, is an important determinant of the observed elastic response and the ability to form physical gels.

Role of viscoelasticity in the tube model of airway reopening. II. Non-Newtonian gels and airway simulation.

The influence of viscoelastic gels as lining fluids on the pressure-velocity relationships in an airway tube model (Gaver et al. J. Appl. Physiol. 69: 74-85, 1990) was examined. A flow instability was observed due to the occurrence of a sol-gel transition in the viscoelastic properties under flow conditions. We further report measurements of the viscoelastic properties of airway secretions. Airway secretions are gels under small strains and have a yield stress of 4-7 dyn/cm2. Secretions from the pharyngeal airway show lower elasticity than secretions from the trachea. The airway reopening process is simulated using a Weibel lung geometry by incorporating the constitutive equations from the model gel studies and utilizing the rheological data on airway secretions. In these simulations, a "popping-open" phenomenon arises from a flow instability in airway generations 8-14 when the rheological properties of the lining fluids are assumed to be similar to those of pharyngeal secretions. On the basis of these studies, the elasticity of airway secretions plays an important role in airway reopening.

Rheological studies of the interaction of mucins with alginate and polyacrylate.

The associative interaction of purified ovine and porcine submaxillary mucins (OSM and PSM) with sodium alginate was evaluated by comparing the rheological properties of mixtures against those of pure alginate and mucin in dilute, semi-dilute, and concentrated solutions. These systems were investigated as models for the interaction of mucin with the extracellular alginate produced by Pseudomonas aeruginosa. In dilute solution, evidence for such interaction cannot be obtained because aggregate species exist both in the OSM-alginate mixtures as well as in pure OSM. However, in the semi-dilute regime, mixtures containing a higher proportion of mucin show systematically higher viscosities than those predicted by simple additivity. In concentrated solutions containing higher proportions of mucin, an enhanced elastic response is observed. These results demonstrate a substantial binding interaction of mucins with alginate. This property is not observed in mixtures containing a high proportion of alginate, suggesting that mucins possess relatively low numbers of interacting sites. Introduction of 3 mM Ca2+ ions to all mucin-alginate mixtures enhances the elasticity due to gelation of alginate. Finally, comparison of the rheological properties of PSM-alginate mixtures with those of PSM-polyacrylate mixtures indicates that the binding strength of alginate to mucin is significantly weaker than that of polyacrylate.

Solvent effects on the viscoelastic behavior of porcine submaxillary mucin.

Rheological methods have been used to investigate the intermolecular interactions of porcine submaxillary mucins (PSM) in solution. PSM is a high molecular weight glycoprotein consisting of a linear, semi-flexible protein backbone to which a large number of oligosaccharides (1-5 saccharide units) are attached as side chains. Concentrated aqueous solutions of PSM containing different amounts of guanidine hydrochloride (GdnHCl) were subjected to both controlled stress and controlled strain rheological analyses. In the absence of GdnHCl, PSM solutions exhibit viscoelastic properties characteristic of a gel: the storage modulus, G', is much larger than the loss modulus, G", at all deformation frequencies, and the compliance is 100% recoverable at small stresses, indicative of strong intermolecular interactions. In 3.0 M aqueous GdnHCl, PSM forms a viscoelastic solution, with G" > G' at all frequencies and a relatively small recoverable compliance, pointing to disruption of the intermolecular interactions by the chaotropic salt. Intermediate behavior is observed in 1.5 M GdnHCl, characteristic of a marginal gel: G' approximately G" and greater than 50% recoverable compliance. In dilute solution, PSM behaves viscoelastically as a typical polyelectrolyte. However, concentrated solutions are turbid, the turbidity decreasing as GdnHCl is added, indicating that extensive intermolecular association accompanies the gelation process. The results show that although PSM is secreted in nature as a viscous solution, it can form gels that are similar to those of tracheobronchial and gastric mucins, and suggest common features to the gelation mechanism, with the strength of the gel correlated with the length of the oligosaccharide side chains.

Viscoelastic properties of human tracheobronchial mucin in aqueous solution.

Human tracheobronchial mucin isolated from cystic fibrosis patients (CF HTBM) was purified using a combination of gel filtration and density gradient centrifugation. The resulting mucin was fractionated to reduce polydispersity and to facilitate studies of the molecular weight dependence of mucin viscoelasticity in concentrated solution. The viscoelastic properties of CF HTBM were examined in distilled water, 0.1M salt solutions and chaotropic solvents. In controlled strain experiments (strain > or = 5%) with increasing mucin concentration, a crossover from sol to gel behavior is observed. The gel strength, as measured by the magnitude of the storage modulus at comparable mucin concentrations, is greatest for distilled water, intermediate for 0.1M NaCl, and lowest for 6M GdnHCl. In distilled water, high molecular weight mucin undergoes a sol-gel transition at approximately 12 mg/mL, and shows evidence of a plateau modulus at higher concentrations. The storage and loss moduli of concentrated high molecular weight fractions in 6M GdnHCl exhibit a power law dependence on frequency typical of weak gels near the sol-gel transition at 20 mg/mL. Similar rheology is observed in 0.1M NaCl and 0.091M NaCl/3 mM CaCl2, but with evidence for additional weak associations at low frequency. The power law exponent in these systems is 0.70 +/- 0.02, in good agreement with prediction for networks formed by a percolation mechanism. Low molecular weight fractions in these solvents exhibit a fluid-like viscoelastic response. However, low molecular weight mucin in distilled water shows a strain-dependent increase in elasticity at low frequency indicative of weak intermolecular associations. Comparison of the rheological behavior of CF HTBM with our earlier studies of ovine submaxillary mucin lends support to the idea that carbohydrate side-chain interactions are important in the gelation mechanism of mucins.

Role of viscoelasticity in tube model of airway reopening. I. Nonnewtonian sols.

This investigation used a previously described bench-top device (Gaver et al., J. Appl. Physiol. 69: 74-85, 1990) to examine the role of nonnewtonian and viscoelastic fluids on events at reopening of a closed flexible tube. Aqueous sodium alginate solutions with and without calcium chloride and sodium dodecyl sulfate in desired concentrations provided fluids with a wide range of surface tensions, storage and loss moduli, and nonnewtonian steady shear viscosity. Dimensionless analysis, using the shear rate-dependent viscosities, was applied to reduce reopening pressure-meniscus velocity data to a master curve. With regard to fluid properties, we found that 1) fluid elasticity strongly changes the pressure-velocity relationship, causing flow instability at higher meniscus velocities; 2) decreasing surface tension gives rise to a smaller yield pressure for reopening; and 3) whereas larger tubes are easier to open, smaller tubes produce additional shear thinning of the lining fluid. These results suggest that, for both the upper (large) and lower (small) airways, nonnewtonian and viscoelastic properties of the mucosal fluid modify the time of closure and rate of reopening.

Early experience with endoscopic carpal tunnel release.

83 hands in 69 patients had endoscopic carpal tunnel release by the Chow method over a 14-month period. 78% of these had a satisfactory outcome with relief of symptoms and no complications. Poor results were explained by incomplete ligament division (five patients), wrong diagnosis (two hands), and post-operative nerve problems (five hands). Two patients after Colles' fractures had unsatisfactory results. Serious complications included two median nerve lacerations. One of these was clearly a result of deviation from the standard protocol. The authors believe that the technique has advantages over open release but they are divided on whether the benefits outweigh the risk of nerve injury.

Viscoelastic studies of extracellular matrix interactions in a model native collagen gel system.

We describe an in vitro test of the hypothesis that viscoelastic properties of the collagen fiber network of skin are influenced by interactions between the macromolecular components in the extracellular matrix. Native type I collagen gels were investigated as a mechanical analog for connective tissue. A series of gels were formed under physiological conditions via fibril precipitation in the presence of selected matrix macromolecules, including dermatan sulfate (DS), hyaluronic acid (HA), dermatan sulfate proteoglycan (DSPG), fibronectin (FN) and elastin. Viscoelastic measurements and transmission electron microscopy were performed to explore the relationship between mechanical strength and fibril morphology. The results demonstrate that associative interactions of DSPG and HA with collagen fibrils, as well as variations in collagen fibril size distribution and the amount of elastin, can modify the viscoelastic behavior of the model collagen gels. Addition of DSPG, DS and HA increases both storage and loss moduli, G' and G"; morphological examination shows adhesive binding of these species to the collagen fibrils. At 37 degrees C, elastin increases G' by forming elastic coacervate particles. FN has no effect on the gel viscoelasticity. The observed effects are discussed in terms of current clinical observations on age-related changes in the mechanical properties of skin.

Viscoelastic behavior of fractionated ovine submaxillary mucins.

Solution properties of fractionated ovine submaxillary mucin (OSM) and asialo OSM (aOSM) in aqueous guanidine hydrochloride have been investigated using light scattering and rheological methods. For the first time we present viscometric evidence in both dilute and concentrated solution that the molecular structure of OSM is that of a wormlike chain. The intrinsic viscosity shows molecular weight dependence consistent with the linear extended chain conformation observed by light scattering measurements. The viscoelastic behavior of the OSM fractions in aqueous guanidine hydrochloride was further examined above the overlap concentration as a function of molecular weight and temperature. Under these solvent conditions in which the role of nonbonding intermolecular interactions is minimized, OSM shows predominantly fluid like behavior. However, high molecular weight OSM shows evidence of the existence of an entanglement network at high concentration. The frequency-dependent shear storage and loss moduli at all concentrations and molecular weights can be scaled to yield a master curve by incorporating typical viscoelastic shift parameters. The entanglement molecular weight and concentration are consistent with literature data for extended, semiflexible wormlike chains. The behavior of aOSM is similar to that of intact OSM at comparable degrees of coil overlap, indicating that the terminal sialic acid residue on the carbohydrate side chain has no effect on the rheology of concentrated OSM solutions beyond that due to an increase in the hydrodynamic volume.