Pan-tissue scaling of stiffness versus fibrillar collagen reflects contractility-driven strain that inhibits fibril degradation.

Polymer network properties such as stiffness often exhibit characteristic power laws in polymer density and other parameters. However, it remains unclear whether diverse animal tissues, composed of many distinct polymers, exhibit such scaling. Here, we examined many diverse tissues from adult mouse and embryonic chick to determine if stiffness ( E tissue ) follows a power law in relation to the most abundant animal protein, Collagen-I, even with molecular perturbations. We quantified fibrillar collagen in intact tissue by second harmonic generation (SHG) imaging and from tissue extracts by mass spectrometry (MS), and collagenase-mediated decreases were also tracked. Pan-tissue power laws for tissue stiffness versus Collagen-I levels measured by SHG or MS exhibit sub-linear scaling that aligns with results from cellularized gels of Collagen-I but not acellular gels. Inhibition of cellular myosin-II based contraction fits the scaling, and combination with inhibitors of matrix metalloproteinases (MMPs) show collagenase activity is strain - not stress- suppressed in tissues, consistent with past studies of gels and fibrils. Beating embryonic hearts and tendons, which differ in both collagen levels and stiffness by >1000-fold, similarly suppressed collagenases at physiological strains of ∼5%, with fiber-orientation regulating degradation. Scaling of E tissue based on 'use-it-or-lose-it' kinetics provides insight into scaling of organ size, microgravity effects, and regeneration processes while suggesting contractility-driven therapeutics.

Cortisol regulates insulin-like growth-factor binding protein (igfbp) gene expression in Atlantic salmon parr.

The growth hormone (Gh)/insulin-like growth-factor (Igf)/Igf binding protein (Igfbp) system regulates growth and osmoregulation in salmonid fishes, but how this system interacts with other endocrine systems is largely unknown. Given the well-documented consequences of mounting a glucocorticoid stress response on growth, we hypothesized that cortisol inhibits anabolic processes by modulating the expression of hepatic igfbp mRNAs. Atlantic salmon (Salmo salar) parr were implanted intraperitoneally with cortisol implants (0, 10, and 40 µg g-1 body weight) and sampled after 3 or 14 days. Cortisol elicited a dose-dependent reduction in specific growth rate (SGR) after 14 days. While plasma Gh and Igf1 levels were unchanged, hepatic igf1 mRNA was diminished and hepatic igfbp1b1 and -1b2 were stimulated by the high cortisol dose. Plasma Igf1 was positively correlated with SGR at 14 days. Hepatic gh receptor (ghr), igfbp1a, -2a, -2b1, and -2b2 levels were not impacted by cortisol. Muscle igf2, but not igf1 or ghr, levels were stimulated at 3 days by the high cortisol dose. As both cortisol and the Gh/Igf axis promote seawater (SW) tolerance, and particular igfbps respond to SW exposure, we also assessed whether cortisol coordinates the expression of branchial igfbps and genes associated with ion transport. Cortisol stimulated branchial igfbp5b2 levels in parallel with Na+/K+-ATPase (NKA) activity and nka-α1b, Na+/K+/2Cl--cotransporter 1 (nkcc1), and cystic fibrosis transmembrane regulator 1 (cftr1) mRNA levels. The collective results indicate that cortisol modulates the growth of juvenile salmon via the regulation of hepatic igfbp1s whereas no clear links between cortisol and branchial igfbps previously shown to be salinity-responsive could be established.

Separation using planar chromatography with electroosmotic flow.

Planar chromatography with electroosmotic flow is used to separate either a mixture of dyes using 80% aqueous ethanol as the mobile phase or a mixture of miscellaneous compounds using 45% aqueous acetonitrile as the mobile phase. Both mobile phases are 1.0 mM in N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid (TAPS) buffer. Separations using this technique are faster and more efficient than the same separations by conventional TLC. The respective relationships between migration velocity and applied potential, and between analysis time and distance migrated, are presented.

Biocompatibility of hemodialysis membranes: evaluation in an ovine model.

We studied the cardiopulmonary, hematologic, and inflammatory response to hemodialysis with seven different membranes in sheep. We also compared acetate dialysate with bicarbonate dialysate and evaluated the role of thromboxane in mediating these responses to dialysis with Cuprophan membranes (Baxter Healthcare Corp., Renal Division, Deerfield, Ill.) in sheep. The data generated in these studies indicate that dialyzer membranes can be divided into three major categories, defined by propensity to activate complement. High complement activators such as Cuprophan (low surface-area CF-1511 and high surface-area ST-25 dialyzers) produced dramatic neutropenia and hypoxemia and significant (p < 0.01) increases in the plasma concentration of thromboxane and in mean pulmonary artery blood pressure. The magnitude of these effects appeared to be surface area related. The low-flux Fresenius F-6 polysulfone membrane (Fresenius USA Inc., Concord, Calif.) also resulted in the generation of significant levels of C3a. In contrast, low complement activators such as polyacrylonitrile (AN-69; Gambro Hospal, Inc., Lakewood, Colo.) and cellulose triacetate (CT-110G; Baxter) produced little or no neutropenia, small transient increases in thromboxane, and no rise in mean pulmonary artery pressure. Dialyzers with intermediate complement-activating potential such as cellulose acetate (CA-110; Baxter) and Hemophan (HT-100; Baxter) produced small to moderate degrees of neutropenia and small increases in thromboxane and mean pulmonary artery pressure. Treatment of sheep with sodium ibuprofen before dialysis with Cuprophan CF-1511 membranes prevented the initial increases in mean pulmonary arterial pressure and thromboxane generation and the decrease in arterial oxygen tension, but did not affect the degree of complement activation or neutropenia. In sheep undergoing Cuprophan dialysis, bicarbonate dialysate did not prevent the increase in circulating complement and the associated neutropenia otherwise seen during the early portions of dialysis with acetate dialysate. Bicarbonate dialysate did, however, reduce (not prevent) the initial increases in thromboxane and mean pulmonary arterial pressure, and the magnitude of the hypoxemia seen with the use of acetate dialysate. The results of these experiments therefore indicate that (1) reactions in sheep correlate well with data collected in human beings and the model can be an effective means for comparing novel dialysis membranes and pharmacologic interventions during dialysis and (2) although complement appears to be the transducer of the hematologic and immunologic response, thromboxane appears to be the final effector of the cardiovascular responses to hemodialysis with Cuprophan membranes.

Wheat germ agglutinin inhibits the C5a receptor interaction: implications for receptor microheterogeneity and ligand binding site.

Wheat germ agglutinin (WGA) has been shown to inhibit the interaction of C5a with the C5a receptor on both polymorphonuclear neutrophils (PMNs) and the histiocytic cell line U937. The level of inhibition with isolated receptor preparations is 100%, and on intact cells 10 to 20% of the receptor population appear to retain their ability to bind C5a in the presence of WGA. In contrast, this lectin completely inhibits the C5a-mediated degranulation of PMN primary and secondary granules, suggesting that the population of C5a receptors responsible for mediating degranulation is also recognized by WGA. More than 50% of the receptors appear to be blocked before an effect on degranulation occurs. This inhibition by WGA does not appear to be due to down-regulation of C5a receptors from the cell surface, excessive aggregation of receptor sites, or interaction of WGA with the carbohydrate portion of the C5a molecule. The inhibition is reversed by N-acetylglucosamine but not by sialic acid. This effect appears to be specific for WGA because various other lectins do not inhibit the C5a receptor interaction. That the inhibition by WGA is due to direct binding of the lectin to N-acetylglucosamine residues on the C5a receptor is strongly supported by the ability of the cross-linked C5a-receptor complex to bind to and be specifically eluted from a WGA-Affigel affinity matrix. These observations are consistent with hypothesis that the population of C5a receptors on leukocytes exhibits microheterogeneity with respect to structure (carbohydrate content) and/or function.

C5a and formyl peptide receptor regulation on human monocytes.

Regulation of C5a and formyl-methionine-leucine-phenylalanine-lysine (fMLPL) receptors on human monocytes has been studied using fluorescein-conjugated derivatives and flow cytometry. Monocytes have receptors for each of these ligands, as evidenced by their ability to bind specifically biologically active fluorescein derivatives of these ligands. Quenching experiments showed that bound fluoresceinated C5a and fMLPL are rapidly internalized at 37 degrees C. Once internalized, monocytes are able to reexpress these receptors, returning to control levels within approximately 90 min. This contrasts with rate differences seen in polymorphonuclear neutrophils (PMNs), where fMLPL receptors return more rapidly (approximately 30 min) than do C5a receptors (approximately 100 min). Monensin inhibited the reexpression of C5a but not fMLPL receptors, suggesting that a receptor recycling process is necessary to replenish C5a receptors on the monocyte surface. Similar although less efficient inhibition of C5a receptor reexpression was observed with NH4Cl treatment. Reexpression of both C5a and fMLPL receptors was independent of extracellular Ca2+. Treatment with various agents known to stimulate monocytes and PMNs increased the expression of fMLPL receptors in both cell types but either had no effect on or reduced the level of C5a receptor expression. This would indicate that monocytes, like PMNs, have intracellular pools of preformed fMLPL receptors, available for reexpression. These studies show that, like PMNs, monocytes modulate C5a and fMLPL receptors through different mechanisms. Furthermore, monocytes are capable of reexpressing these receptors following exposure to ligand, a theoretical requirement for chemotaxis.

Modulation of monocyte chemotactic function in inflammatory lesions. Role of inflammatory mediators.

Monocyte recruitment and accumulation in the synovial tissue is pivotal in the evolution of rheumatoid arthritis (RA). In the present study we examined the chemotactic potential of monocytes obtained from synovial fluid (SF) of patients with RA. Functionally, SF monocytes exhibited greatly diminished chemotactic activity to C5a compared with monocytes from the peripheral blood. In contrast, their chemotactic responsiveness to the synthetic peptide, FMLP, was nearly normal. To define a mechanism for this differential chemotactic dysfunction, cell-surface receptors for C5a (C5aR) and FMLP (FMLP-R) were evaluated. Whereas FMLP-R expression was similar on both blood and inflammatory monocytes, C5aR expression was markedly reduced on SF cells. Because decreased C5a binding in certain RA SF samples could not be attributed to free C5a, known or suspected components of inflammatory SF were evaluated for their ability to modulate chemotactic ligand receptors. Bacterial products including LPS and streptococcal cell walls, which are potent monocyte activators, down-regulated C5aR without affecting FMLP-R. Moreover, the cytokines IFN-gamma and granulocyte-macrophage-CSF selectively decreased C5aR in parallel with decreased in vitro chemotactic activity to C5a. Thus, these data indicate that 1) synovial effusions may contain C5a and/or inflammatory mediators that modulate phenotypic and functional changes in monocytes, 2) chemotactic ligand receptors are independently regulated in inflammatory lesions, and 3) decreased C5aR expression and chemotactic potential likely provide a mechanism whereby monocyte-macrophages persist within the inflamed synovium.

IFN-gamma inhibits inflammatory cell recruitment and the evolution of bacterial cell wall-induced arthritis.

Localization of streptococcal cell wall Ag (SCW) in the synovial tissue of treated rats induces an influx of leukocytes and a cell-mediated immune response leading to arthritis and joint destruction. Systemic administration of the T cell product, IFN-gamma (10(6) U/kg/day), suppresses the recruitment of leukocytes into the synovium and effectively inhibits the inflammation and pathology characteristic of SCW-induced arthritis (articular index 10.4 +/- 0.6 for SCW vs 2.0 +/- 0.7 for SCW with IFN-gamma, p less than 0.005). Monocyte-macrophages from animals treated with IFN-gamma exhibited defective chemotactic responses when tested in vitro and furthermore, monocytes cultured with IFN-gamma (25 to 500 U/ml) in vitro had significantly suppressed chemotactic responses to the complement fragment C5a (p less than 0.005). The decreased ability to migrate to C5a was associated with decreased binding of fluorochrome-conjugated C5a indicative of reduced expression of C5a receptors. Based on these data, IFN-gamma that induces monocyte maturation as reflected by increased Ia expression conversely inhibits C5a receptor expression. Although locally elevated IFN-gamma levels may serve to inhibit recruitment away from an inflammatory site, systemic exposure to IFN-gamma appears to inhibit leukocyte recruitment to the inflammatory site by its ability to induce premature maturation and concomitant inability to respond to certain chemotactic ligands. Inasmuch as monocyte recruitment to the synovium is pivotal in the development of SCW-induced polyarthritis, the ability of IFN-gamma to inhibit this event effectively inhibits the synovial pathology.