Hematogenous infection with the yeast Candida albicans now occurs with increasing frequency in the neonate, the immunocompromised patient, and the hyperglycemic or hyperalimented host. Yeast-phase C. albicans expresses a protein that is antigenically and structurally related to CD11b/CD18, a member of the 2 integrins and a well-characterized adhesin for mammalian neutrophils. Both the neutrophil protein and its analogue in C. albicans have an identical affinity for the C3 ligand iC3b, and both proteins are significantly increased in expression at 370C. Given these several similarities, we therefore studied the role of the integrin analogue on C. albicans in the adhesion of the yeast to human umbilical vein endothelium (HUVE).After growth of C. albicans in 20 mM D-glucose, as opposed to 20 mM L-glutamate, flow cytometric analysis with monoclonal antibodies recognizing the a-subunit of CDMMb/ CD18 demonstrated a 25.0% increase in mean channel fluorescence (range 18.4-31.8%), as well as an increased percentage of yeasts fluorescing (P < 0.02). This increased intensity of fluorescence, which corresponds to increased expression of the integrin analogue, also correlated with a significant increase of 30-80% in adhesion of glucose-grown C. albicans to HUVE (P < 0.02). Blockade of the integrin analogue on C. albicans by monoclonal antibodies recognizing adhesive epitopes on neutrophil CD11b/CD18 inhibited glucose-enhanced adhesion of C. albicans to HUVE. Incubation of glucose-grown C. albicans with saturating concentrations of purified human iC3b, the ligand for CD1lb/CD18, reduced adhesion of the yeast to HUVE by 49.7%, whereas BSA in equimolar concentration had no effect (P < 0.001). These results identify a glucose-responsive integrin analogue on C. albicans as one of possibly several cellular structures that mediate adhesion of the yeast to human endothelium. (J. Clin. Invest. 1991. 87:1896-1902

Previous findings suggest that neuroadaptations downstream of D-1 dopamine (DA) receptor stimulation in nucleus accumbens (NAc) are involved in the enhancement of drug reward by chronic food restriction (FR). Given the high co-expression of D-1 and GluR1 AMPA receptors in NAc, and the regulation of GluR1 channel conductance and trafficking by D-1-linked intracellular signaling cascades, the present study examined effects of the D-1 agonist, SKF-82958, on NAc GluR1 phosphorylation, intracranial electrical self-stimulation reward (ICSS), and reversibility of reward effects by a polyamine GluR1 antagonist, 1-NA-spermine, in ad libitum fed (AL) and FR rats. Systemically administered SKF-82958, or brief ingestion of a 10% sucrose solution, increased NAc GluR1 phosphorylation on Ser845, but not Ser831, with a greater effect in FR than AL rats. Microinjection of SKF-82958 in NAc shell produced a reward-potentiating effect that was greater in FR than AL rats, and was reversed by co-injection of 1-NA-spermine. GluR1 abundance in whole cell and synaptosomal fractions of NAc did not differ between feeding groups, and microinjection of AMPA, while affecting ICSS, did not exert greater effects in FR than AL rats. These results suggest a role of NAc GluR1 in the reward-potentiating effect of D-1 DA receptor stimulation and its enhancement by FR. Moreover, GluR1 involvement appears to occur downstream of D-1 DA receptor stimulation rather than reflecting a basal increase in GluR1 expression or function. Based on evidence that phosphorylation of GluR1 on Ser845 primes synaptic strengthening, the present results may reflect a mechanism via which FR normally facilitates reward-related learning to re-align instrumental behavior with environmental contingencies under the pressure of negative energy balance.

During inflammation polymorphonuclear cells (PMNs) are exposed to agonistic stimuli including activated complement, kallikrein, arachidonic acid metabolites, monokines, and platelet-activating factor (PAF). We report that PAF not only directly activates PMNs but in miniscule quantities (10(-12) mol/L) “primes” them as well, that is, permits PMNs to respond to subsequent stimuli that would be otherwise ineffectual. PAF priming of responses including superoxide generation, elastase release, and aggregation is time dependent and is maximal within five minutes. PAF need not be present during the subsequent exhibition of PMN agonists, but priming is inhibited by cold and is also inhibited by the PAF receptor antagonists BN 52021, L-652, and kadsurenone. An intact PAF molecule is required because lyso-PAF and methoxy-PAF do not prime PMN responses. PAF priming is associated with both enhanced expression of the adhesive glycoprotein identified by OKM- 1 antibody and an enhanced rise in intracellular calcium levels in response to the subsequent addition of agonists such as FMLP. PMNs primed with PAF and stimulated with either F-Met-Leu-Phe or phorbol esters are more effective in lysing and detaching cultured human endothelial cells--damage that can also be inhibited by the PAF antagonists. Because PAF is synthesized and exhibited on surfaces of endothelial cells perturbed by coagulation, we suggest that this lipid may potentiate otherwise trivial activators of marginated PMNs so that they become damaging to the PAF-synthesizing endothelium itself. If so, our studies suggest a possible therapeutic role for PAF inhibitors in excessive inflammatory states.

We have shown previously that fluid phase platelet-activating factor (PAF) can enhance or "prime" polymorphonuclear (PMN) responses to subsequent stimulation with agonists such as formyl-methionine-leucine-phenylalanine (FMLP). Since thrombin induces PAF production in endothelial cells, we tested whether this thrombin-provoked endothelial PAF primes responses of marginated PMNs. Monolayers of human umbilical vein endothelial cells were exposed to either thrombin (0.5-5.0 units/ml) or buffer for up to 5 min and then PMNs were layered on top of the endothelial cells. After a further 5 min incubation, the PMNs were stimulated with a suboptimal concentration of FMLP (10(-7) M), and their superoxide production, elastase release, adhesion to endothelium, and capacity to cause endothelial cell lysis and detachment were assessed. Thrombin pretreatment significantly enhanced each of these FMLP-stimulated neutrophil responses. The extent of this enhancement correlated with both the dose and duration of thrombin treatment of endothelial cells and also the duration of PMN incubation with thrombin-exposed endothelium. Evidence that the augmentation was due to endothelial-derived PAF was obtained as follows: (1) thrombin induced [3H]acetate incorporation into endothelial PAF (assayed in lipid extracts); (2) antithrombin III conjointly inhibited this [3H]acetate uptake and prevented the priming effect of thrombin-treated endothelium on PMN responses; and (3) the PAF receptor antagonist BN52021, when preincubated with PMNs, also effectively blocked the enhancement of PMN responses. We conclude that thrombin stimulation of endothelial cells initiates a sequence of events culminating in the production of PAF--a membrane phospholipid capable of priming marginated PMNs. We suggest that this coagulation-fostered endothelial/PMN interaction may underlie a paracrine response that may potentiate PMN-mediated endothelial injury during sepsis and other thrombin-generating disorders.

An increase in intracellular calcium level is an important signal in the regulation of cellular responses under normal and pathological conditions. Because two key enzymes in the synthetic pathway of platelet activating factor (PAF), phospholipase A2 and acetyltransferase, are calcium dependent, we hypothesized that calcium channel blockade may inhibit agonist-induced PAF synthesis. Primary cultures of human umbilical vein endothelial cells (EC), pre-incubated with [3H]acetate, were exposed to thrombin (5 U/mL) and PAF production was quantitated by incorporation of radiolabel into the EC lipid fraction co-migrating with exogenous PAF in thin-layer chromatography. The effect of pre-incubation with calcium channel blockers (verapamil, diltiazem, 10(-4) M) or buffer was determined. Results (triplicate experiments, * P less than 0.05 vs buffer, P less than 0.05 vs thrombin) demonstrate that pre-incubation with calcium channel blocker markedly inhibits thrombin-induced PAF production (verapamil:buffer 273 +/- 122, thrombin 10,735 +/- 1524*, thrombin + verapamil 178 +/- 91 cpm/plate; diltiazem:buffer 1097 +/- 581, thrombin 15,283 +/- 2661*, thrombin + diltiazem 280 +/- 56 cpm/plate). The effect of diltiazem was dose-dependent (% inhibition: 10(-7) M, 46%; 10(-5) M, 60%; 10(-4) M, 98%). Diltiazem also inhibited bradykinin (10(-8) M) induced PAF synthesis. In calcium-free medium or in the presence of LaCl3 (10(-3) M), the PAF response of EC to thrombin was blunted (buffer 582 +/- 360, thrombin 5394 +/- 1069, thrombin + calcium free medium 1055 +/- 571, thrombin + LaCl3 1271 +/- 58 cpm/plate). We conclude that calcium channel blockers prevent agonist-induced PAF synthesis, possibly by preventing cellular calcium influx and activation of PAF synthetic enzymes. We speculate that this mechanism may underlie, at least in part, the beneficial effect of calcium channel blockade under various pathological conditions.