Leukocytes respond to lipopolysaccharide (LPS) at nanogram per milliliter concentrations with secretion of cytokines such as tumor necrosis factor-alpha (TNF-alpha). Excess secretion of TNF-alpha causes endotoxic shock, an often fatal complication of infection. LPS in the bloodstream rapidly binds to the serum protein, lipopolysaccharide binding protein (LBP), and cellular responses to physiological levels of LPS are dependent on LBP. CD14, a differentiation antigen of monocytes, was found to bind complexes of LPS and LBP, and blockade of CD14 with monoclonal antibodies prevented synthesis of TNF-alpha by whole blood incubated with LPS. Thus, LPS may induce responses by interacting with a soluble binding protein in serum that then binds the cell surface protein CD14.

SummaryTumor necrosis factor a, granulocyte colony-stimulating factor, granulocyte/macrophage colonystimulating factor, and formyl peptide were each found to cause a twofold increase in expression of CD14 on the surface of polymorphonuclear leukocytes (PMN). Upregulation of CD14 was complete by 20 min and thus appeared to result from expression of preformed stores ofprotein . The CD14 on the surface ofPMN was shown to serve two biological functions. It bound particles coated with complexes oflipopolysaccharide (LPS) and LPS binding protein (LBP). This binding activity was enhanced by agonists that upregulated CD14 expression and may serve in the clearance of Gram-negative bacteria opsonized with LBP. Interaction of CD14 with LPS in the presence of LBP or serum also caused a dramatic, transient increase in the adhesive activity of CR3 (CD11b/CD18) on PMN . Enhanced activity of CR3 and other members of the CD11/CD18 family underlies many of the known physiological responses ofPMN to LPS and may be a central feature of the in vivo responses of PMN to endotoxin. BacterialLPS(endotoxin) is known to have profound physiological effects on PMN both in vivo and in vitro. Animals respond to intravenous LPS with a rapid fall in the number of circulating PMN and a concomitant accumulation ofPMN in the lungs (1) . Isolated PMN respond to LPS with increased adhesion to protein-coated surfaces (2), enhanced ability to mount an oxidative burst (3), and enhanced microbicidal powers (4) . Many of these responses to LPS appear to depend at least in part on enhanced function of the three members of the CD11/CD18 family of adhesion-promoting receptors (LFA1, CR3, and p150,95, also known as the 02 or leukocyte integrins) on the surface of PMN . Adhesion of PMN to endothelium requires the participation ofCDII/CD18 molecules (5-7), and blockade ofCD18 with mAbs prevents accumulation ofPMN in the lungs ofendotoxin-treated animals (8) . Adhesion ofPMN to protein-coated glass (5), enhanced oxidative burst in response to soluble agonists (9), and microbicidal activity (5) are also dependent on increased CD11/CD18 function. Here, we directly measure the effect of LPS on the adhesive function ofCR3 (CD11b/CD18) and describe a dramatic enhancement of its adhesive activity by complexes of LPS with proteins from the serum.Recent studies have described a novel mechanism by which mononuclear cells may respond to LPS (10, 11) . LPS is first bound by the serum protein LPS binding protein (LBP)l, and the resulting LPS-LBP complex is then recognized by CD14, a 55-kD glycoprotein that is strongly expressed on monocytes and macrophages. LBP and CD14 serve two physiological roles. These proteins act as opsonin and opsonic receptor, respectively, to promote the phagocytic uptake of bacteria or LPS-coated particles by macrophages (12). They also dramatically enhance the ability of mononuclear cells to synthesize TNF in response to endotoxin . Addition of LBP speeds the synthesis of TNF and enables a response to doses of LPS 100-fold lower than are otherwi...

The classical opsonins C3 and IgG bind to microorganisms and facilitate phagocytosis. Recent work suggests that additional proteins may also have opsonic activity. The acute-phase reactant, C-reactive protein (CRP), binds to phosphocholine residues on gram-positive bacteria and facilitates their interaction with phagocytes (1) . Another acute-phase reactant, mannose binding protein (MBP),' binds to mannose residues on bacteria and fungi and facilitates recognition by phagocytes (2). We have investigated a third recently discovered acute-phase reactant, lipopolysaccharide binding protein (LBP) (3) . LBP is a 60-kD glycoprotein present at concentrations <100 ng/ml in the serum of healthy rabbits . During the acute phase, LBP is synthesized by hepatocytes (Ramadori, G., K.-H. Meyer zum Buschenfelde, P S. Tobias, J. C . Mathison, and R. J . Ulevitch, manuscript submitted for publication), and reaches concentrations of 30-50 Fig/ml in serum . Here we show that LBP binds to LPS on the surface of bacteria and to LPS inserted into erythrocyte membranes . Binding of LBP to these particles dramatically enhances their interaction with macrophages . Particle-bound LBP is recognized by a receptor that is mobile in the plane of the membrane and that appears distinct from other opsonic receptors .

SummaryTwo classes of adhesion molecules have well-defined roles in the attachment of unstimulated polymorphonuclear leukocytes (PMN) to cytokine-treated endothelial cells. Endothelial-leukocyte adhesion molecule 1(ELAM-1) on endothelial cells interacts with specific carbohydrate residues on the PMN, and the leukocyte integrins (CD18 antigens) on PMN interact with intracellular adhesion molecule 1 and other structures on endothelium . Here we show that these two classes of molecules can act sequentially in an "adhesion cascade". Interaction of PMN with SLAM-1-bearing endothelial cells causes PMN to express enhanced adhesive activity of the integrin CR3 (CD11b/CD18) . Expression of ELAM-1 on the cytokine-treated endothelium appears both necessary and sufficient for the stimulation of CR3 activity since blockade of ELAM-1 with mAbs prevents the activation of CR3 by cytokine-treated endothelium, and immobilized recombinant ELAM-1 activates CR3 . The ability to activate CR3 is shared by chemattractants, suggesting that ELAM-1 may serve as a "tethered chemattractants" This hypothesis is strengthened by the observation that recombinant soluble ELAM-1 directs movement of PMN in chemotaxis chambers. These results suggest a mechanism by which multiple adhesive molecules may function together in diapedesis. ELAM-1 serves both as an adhesin and as a trigger that recruits the participation of additional adhesion molecules. Our results also suggest that ligands for adhesion molecules may also be "receptors" capable of generating intracellular signals.

Bell Communications ResearchThis article describes a 4-year research effort to develop and apply a "user-friendly" method for estimating the standard deviation of job performance in dollars that might permit wider application of utility analysis to personnel activities. The method builds on traditional industrial psychological principles of job analysis and performance measurement, and it allows translation of behaviorally based performance rating data into economic terms. It was applied in a field study of 602 first-level managers, promoted either via a panel selection/interview process or via an assessment center (a) to investigate differences in the mean and variability of dollar-valued job performance and (b) as one input to a comparative utility analysis of the two procedures. Results indicated that the method is feasible, practical, and simple to use. The comparative utility analysis results indicated a significant payoff in terms of improved performance per person per year for the assessment center.Reviews of historical developments in utility analysis (Cascio, 1982;Landy, Farr, & Jacobs, 1982;Schmidt, Hunter, McKenzie, & Muldrow, 1979) have emphasized consistently that the major stumbling block to more widespread use of continuous variable utility analysis (Brogden, 1949;Cronbach & Gleser, 1965) is an inability to develop workable, defensible methods for estimating the dollar value of job performance. It is the variability in job performance that is essential, because if all applicants have roughly the same predicted absolute level of productivity, the development and use of costly, incrementally valid selection procedures are unnecessary. It is important to point out, however, that if the selection ratio and the standard deviation of job performance in dollars are held constant, then utility is a function of validity (Brogden, 1946). Typically there is considerable variability in performance, and, therefore, the benefits of more valid, and perhaps more costly, selection procedures may far outweigh their costs. The purpose of the present study is to report the results of a 4-year research effort to develop and apply a new method for estimating the variability of job performance in dollars that might permit widespread application of utility analysis to personnel activities.Originally we planned simply to tap a sophisticated accounting system that was already in place in order to calculate, for each manager, the cost to the company of having his or her services and the dollar benefits of the output produced. The difference between benefits and costs would yield a net dollar benefit (or loss) to the firm. Over all managers in the study, a mean and The authors would like to express their deep appreciation to two anonymous, hard-working reviewers who provided expert criticism and guidance throughout all sections of this article. We also would like to acknowledge the support and helpful comments, from the very inception of this research, of Joseph L. Moses of AT&T.

Insulin-like growth factor

Abrogation of p53 function occurs in almost all human cancers, with more than 50% of cancers harboring inactivating mutations in p53 itself. Mutation of p53 is indicative of highly aggressive cancers and poor prognosis. The vast majority of mutations in p53 occur in its core DNA binding domain (DBD) and result in inactivation of p53 by reducing its thermodynamic stability at physiological temperature. Here, we report a small molecule, SCH529074, that binds specifically to the p53 DBD in a saturable manner with an affinity of 1-2 M. Binding restores wild type function to many oncogenic mutant forms of p53. This small molecule reactivates mutant p53 by acting as a chaperone, in a manner similar to that previously reported for the peptide CDB3. Binding of SCH529074 to the p53 DBD is specifically displaced by an oligonucleotide with a sequence derived from the p53-response element. In addition to reactivating mutant p53, SCH529074 binding inhibits ubiquitination of p53 by HDM2. We have also developed a novel variant of p53 by changing a single amino acid in the core domain of p53 (N268R), which abolishes binding of SCH529074. This amino acid change also inhibits HDM2-mediated ubiquitination of p53. Our novel findings indicate that through its interaction with p53 DBD, SCH529074 restores DNA binding activity to mutant p53 and inhibits HDM2-mediated ubiquitination.

SnmmJaryWe have previously reported that lipopolysaccharide (LPS) binding protein (LBP) opsonizes endotoxin (LPS) for recognition by CD14 on phagocytes. Here we show that normal human plasma contains high titers of an activity that also binds LPS (Re, 595) and mediates recognition by CD14. Opsonization of LPS-coated particles with plasma enables the particles to be bound by phagocytes. Further, opsonization with plasma also enables subnanogram-per-milliliter concentrations of LPS to induce dramatic alterations in the function of leukocyte integrins on polymorphonudear leukocytes and to induce secretion of tumor necrosis factor by monocytes, suggesting that opsonization by factors in plasma may be important in responses of cells to endotoxin. The opsonic activity in plasma appears distinct from LBP since it is not blocked by neutralizing antibodies against LBP. Surprisingly, the opsonic activity of plasma is not present in a single protein species, but at least two species must be combined to observe activity. Further, the opsonic activity of plasma for LPS is blocked by addition of protease inhibitors, suggesting that proteolytic activity or activities are required for opsonization. These properties are suggestive of the action of a protease cascade, but opsonic activity of plasma is not affected by blockade or depletion of either the complement or dotting cascades. We propose the name "septin" to describe this novel LPS-opsonizing activity in plasma.ukocytes recognize bacterial LPS (endotoxin) through several mechanisms (1). CD18 antigens (2) and the scavenger receptors (3) on leukocytes have been shown to bind particulate and dispersed LPS, respectively. This binding may result in uptake and degradation of LPS but is not associated with stimulation of the ceUs (3, 4). We have recently described a different means of recognizing LPS in which a soluble protein, rather than a membrane-bound molecule, performs the initial binding to LPS. LPS binding protein (LBP) 1 binds to both dispersions of LPS (5) and to LPS on the surface of particles (6). The resulting LPS/LBP complexes are recognized by the receptor CD14 on PMN (7), monocytes, and macrophages (MO) (8). "Opsonization" of bacteria with LBP enables phagocytes to bind and phagncytose the particles (6) in a CD14-dependent fashion (S. D. Wright, unpublished observations), thus resulting in clearance of LPS.CD14 may also initiate the dramatic responses of leukocytes to picogram-per-milliter concentrations of endotoxin. PMN show the most rapid responses with priming for enhanced oxidative activity (9) and enhanced activity of leukocyte integrins (7) observed after 15-30 min of exposure to LPS. Both of these responses of PMN require LBP (7, 10) or serum (7, 11), and both can be blocked by anti-CD14 mAbs (7, and M. J. Pabst, personal communication). Monocytes and MO respond to LPS by synthesizing cytokines such as TNF, Ibl, and IL-6 within 1-2 h. TNF production by rabbit MO in response to sub nanogram-per-milliliter doses of LPS requires LBP (12), and blockade of CD14...