The relationship between alloimmunization and posttransfusion granulocyte survival: experience in a chronic granulomatous disease cohort.

BACKGROUND: The efficacy of granulocyte transfusions in patients with HLA alloimmunization is uncertain. A flow cytometric assay using dihydrorhodamine 123 (DHR), a marker for cellular NADPH oxidase activity, was used to monitor the differential survival of transfused oxidase-positive granulocytes in alloimmunized patients with chronic granulomatous disease (CGD). STUDY DESIGN AND METHODS: Ten patients with CGD and serious infections were treated with daily granulocyte transfusions derived from steroid and granulocyte-colony-stimulating factor-stimulated donors. The proportion of neutrophils with intact oxidase activity was quantitated by DHR fluorescence on samples drawn before and 1 hour after transfusion. The incidence of acute transfusion reactions was correlated with the results of DHR fluorescence and biweekly HLA serologic screening assays. RESULTS: Eight of 10 patients experienced acute adverse reactions in association with granulocyte transfusions. Four had only chills and/or fever, and four experienced respiratory compromise; all eight exhibited HLA alloimmunization. Mean (± SD) oxidase-positive cell recovery was 19.7 ± 17.4% (n = 15 transfusions) versus 0.95 ± 1.59% (n = 16) in the absence and presence of HLA allosensitization, respectively (p < 0.01). Greater than 1% in vivo recovery of DHR-enhancing donor granulocytes was strongly correlated with lack of HLA alloimmunization. CONCLUSION: The ability to detect DHR-positive donor granulocytes by flow cytometry is strongly correlated with absence of HLA alloimmunization and lack of acute reactions to granulocyte transfusions in patients with CGD. If HLA antibodies are present and the survival of donor granulocytes is low by DHR analysis, transfusions should be discontinued, avoiding a therapy associated with high risk and unclear benefit.

Effect of substituted arginine compounds on superoxide production in the rabbit aorta.

Superoxide anion (O2-) blocks the vascular effect of endothelium-derived relaxing factor (EDRF). Previous reports implicate L-arginine or N alpha-substituted arginine compounds as precursors of EDRF. Employing a microassay for the measurement of O2- production by rabbit aortic rings, which is based on the established method of reduction of cytochrome c by O2-, we studied the interaction between O2- and EDRF using L-arginine, an N-substituted arginine compound, namely, N alpha-benzoyl-L-arginine ethyl ester (BAEE), sodium nitroprusside and NG-monomethyl-L-arginine (NMMA), a putative specific inhibitor of EDRF synthesis. Measurements of O2- production were made under basal conditions and upon stimulation with alloxan, the diabetogenic, O(2-)-generating compound. Both BAEE, an EDRF-generating agent (0.3 and 3.0 mM) and sodium nitroprusside, an NO-generating compound (1.7 and 3.4 microM), significantly reduced alloxan-stimulated O2- production, but L-arginine (0.6-3.0 mM) paradoxically increased O2- generation. NMMA (1.0 mM) blocked the inhibitory effect of BAEE on O2- production in an endothelium-dependent manner. Because NMMA is an inhibitor of EDRF, these results suggest that BAEE, but not L-arginine, reduces O2- produced by the endothelium of the rabbit aorta through a mechanism involving EDRF generation.

Superoxide production in the isolated rabbit aorta and the effect of alloxan, indomethacin and nitrovasodilators.

The purpose of this investigation was to develop a simple model to assess superoxide production from isolated vessels and to use this model to study the effects of various compounds on superoxide generation. The established method of cytochrome C reduction by superoxide was modified to measure superoxide production in vascular rings from rabbit aortae. The diabetogenic compound alloxan significantly increased superoxide production in a concentration-dependent manner. The nitrovasodilators nitroprusside and minoxidil exhibited contrasting effects. Nitroprusside inhibited alloxan-stimulated production of superoxide, but minoxidil had no effect, suggesting different mechanisms of action for these drugs. The cyclooxygenase inhibitor indomethacin had no effect on the production of superoxide stimulated by alloxan, demonstrating that superoxide production induced by this compound is not affected by mechanisms involving cyclooxygenase. These data demonstrate the use of a simple, rapid and inexpensive method for measuring superoxide produced by intact vessels. This may be useful in testing drugs exhibiting antioxidant and vasoactive properties. Finally, because superoxide is implicated in the destruction of endothelium-derived relaxing factor, and the presence of the vasodilator nitroprusside reduces superoxide production, it is concluded that some nitrovasodilators may have additional vascular effects through the suppression of superoxide formation.

Constriction of canine coronary arteries by platelet activating factor after brief ischemia.

Platelet activating factor (PAF) has been suggested as a mediator of coronary spasm and acute myocardial ischemia. However, PAF can have either vasodilator or vasoconstrictor activities according to various reports. Because of the importance of endothelium in regulating vascular tone, we hypothesized that changes in endothelial function could modulate some of the observed differences in the activities of PAF. To test this hypothesis, PAF was infused directly into the left anterior descending coronary artery (LAD) of dogs at a rate of 0.3 microgram/min before and after 20 min of LAD ligation followed by reperfusion. Coronary blood flow (CBF) was measured continuously via a Doppler flow probe. Likewise, responses to the endothelium dependent vasodilator, acetylcholine (ACh) were measured before and after the LAD ligation. Before ligation, PAF produced a vasodilatory response in the LAD, resulting in 28.4 +/- 11.0% increase in CBF and a 21.5 +/- 5.8% decrease in coronary vascular resistance (CVR). However, after ligation and subsequent reperfusion, 0.3 microgram/min PAF produced vasoconstriction, resulting in a 10.2 +/- 8.7% decrease in CBF (p < 0.01 compared to pre-infusion change in (CBF), and a 27.8 +/- 23.2% increase in CVR (p = 0.05) compared to pre-infusion change in CVR). The vasodilator response to ACh was markedly blunted by ischemia. These results suggest that the coronary vascular response to PAF may depend upon the functional integrity of the endothelium, with endothelial damage resulting in constrictor responses to PAF.(ABSTRACT TRUNCATED AT 250 WORDS)

Brief ischemia-reperfusion induces stunning of endothelium in canine coronary artery.

BACKGROUND: Brief ischemic episodes that induce stunning of the myocardium may also induce stunning of the coronary endothelium. To test this hypothesis, we examined both in vivo and in vitro responses of canine coronary arteries exposed to brief ischemia. METHODS AND RESULTS: Functional recovery of the endothelium was examined in vivo during reperfusion after 15 minutes of ischemia. Vasodilatory responses to acetylcholine were severely impaired during the first hour of reperfusion but gradually improved over a 90-minute period after ischemia. The vasoconstrictive response to U46619 was enhanced for the first 30 minutes of reperfusion and returned to normal within 60 minutes. In vitro vasomotor responses to potassium chloride, acetylcholine, bradykinin, and sodium nitroprusside were examined in isolated segments of canine coronary arteries preexposed in vivo to brief ischemia (10-30 minutes) and 20 minutes of reperfusion. The results showed enhanced contractile responses and blunted endothelium-dependent but not endothelium-independent vasodilatory responses of arterial rings subjected to 10 minutes of ischemia. Twenty and 30 minutes of ischemia completely impaired endothelium-dependent vasodilation. When reperfusion was extended to 120 minutes after 15 minutes of ischemia, vasodilatory responses to acetylcholine had recovered by almost 90%. Examination of endothelial integrity by transmission electron microscopy after 10-15 minutes of ischemia revealed no evidence of structural damage. Twenty and 30 minutes of ischemia induced cytoplasmic vacuolation, partial detachment of endothelium, and swelling of cytoplasmic organelles. CONCLUSIONS: These data support the hypothesis that brief ischemia-reperfusion induces stunning of endothelium in which endothelium-dependent vasodilatory function is impaired temporarily without morphological damage.