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A radiochromatographic method has been set up to assay the activity of N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD), based on reversed-phase high-performance liquid chromatography (HPLC) and online scintillation counting. The anandamide (N-arachidonoylethanolamine, AEA), product released by NAPE-PLD from the N-arachidonoyl-phosphatidylethanolamine (NArPE) substrate, was separated using a C18 column eluted with methanol-water-acetic acid and was quantified with an external standard method. Baseline separation of AEA and NArPE was completed in less than 15 min, with a detection limit of 0.5 fmol AEA at a signal-to-noise ratio of 4:1. The sensitivity and accuracy of the radiochromatographic procedure allowed detection and characterization of NAPE-PLD activity in very tiny tissue samples or in samples where the enzymatic activity is very low. With this method, we could determine the kinetic constants (i.e., apparent Michaelis-Menten constant (Km) of 40.0+/-5.6 microM and maximum velocity (Vmax) of 22.2+/-3.5 pmol/min per milligram protein toward NArPE) and the distribution of NAPE-PLD activity in brain areas and peripheral tissues of mouse. In addition, we could collect unprecedented evidence that compounds widely used in studies of the endocannabinoid system (e.g., AEA and congeners, receptor a(nta)gonists and inhibitors of AEA degradation) can also affect NAPE-PLD activity.

Studies comparing transfusion and nontransfusion patients suggest an increased risk of postoperative infections in transfusion groups. Supernatants of blood components have been shown to affect the function of T lymphocytes and natural killer cells. Here, we found that supernatants from stored red blood cells (RBCs) inhibit human neutrophil migration in response to formyl peptides and stimulate neutrophil locomotion. These effects can be observed with high dilutions of RBC supernatants, such as 1:5 x 106 (vol/vol), able to trigger locomotion as well as desensitization of the cells to alternative chemoattractants. The phenomenon might be mediated by chemoattractants present in the supernatants. As RBC supernatants failed to mobilize intracellular free calcium, the chemoattractants should belong to the group of pure chemoattractants, that is, soluble Fas ligand (sFasL) and transforming growth factor-beta1 (TGF-beta1), known to act without increasing calcium levels. Recombinant TGF-beta1, but not sFasL, was found to reproduce the ability of RBC supernatants to both inhibit neutrophil response to formyl peptides and stimulate neutrophil locomotion. Moreover, TGF-beta1-immunodepleted supernatants did not display neutrophil-directed activities. Finally, RBC supernatants from RBCs stored after depletion of leukocytes were incapable of affecting neutrophil function. With neutrophils acting as a first-line antimicrobial defense, the ability, shown here, of high dilutions of RBC supernatants to inhibit neutrophil chemotaxis through TGF-beta1 may be a relevant determinant of infections in the postoperative period for transfusion patients. Consistently, the neutrophil chemotactic response to formyl peptide was inhibited by the plasma obtained from 5 transfusion patients.

The immunomodulatory effect of allogeneic blood transfusions (ABT) has been known for many years. However, a complete understanding of the effects of ABT on the recipient's immune system has remained elusive. Soluble HLA class I (sHLA-I), HLA class II (sHLA-II), and Fas ligand (sFasL) molecules may play immunoregulatory roles. We determined by double-determinant immunoenzymatic assay (DDIA) sHLA-I, sHLA-II, and sFasL concentrations in different blood components. sHLA-I and sFasL levels in red blood cells (RBCs) stored for up to 30 days and in random-donor platelets are significantly (P <.001) higher than in other blood components and their amount is proportionate to the number of residual donor leukocytes and to the length of storage. Blood components with high sHLA-I and sFasL levels play immunoregulatory roles in vitro as in allogeneic mixed lymphocyte responses (MLR) and antigen-specific cytotoxic T-cell (CTL) activity, and induce apoptosis in Fas-positive cells. These data suggest that soluble molecules in blood components are functional. If these results are paralleled in vivo, they should be taken into account in transfusion practice. Blood components that can cause immunosuppression should be chosen to induce transplantation tolerance, whereas blood components that lack immunosuppressive effects should be preferred to reduce the risk of postoperative complications and cancer recurrence.