Mark P. Fletcher

Limitation of myocardial infarct size in pigs with a dual lipoxygenase-cyclooxygenase blocking agent by inhibition of neutrophil activity without reduction of neutrophil migration

OBJECTIVES The purpose of this study was to assess the effect of the dual cyclooxygenase-lipoxygenase blocking agent BW755C on the extent of myocardial infarction in the pig and to identify the mechanisms of any cardioprotective action of this drug. BACKGROUND Activated neutrophils contribute to reperfusion injury after myocardial infarction and inhibition of neutrophil function can limit infarct size. METHODS In 9 control and 10 study pigs pretreated with intravenous BW755C (10 mg/kg body weight) 30 min before coronary occlusion, ischemia was induced by a 50-min occlusion of the mid-left anterior descending coronary artery, followed by 3 h of reperfusion. Heart rate, arterial pressure, left ventricular end-diastolic pressure, the first derivative of left ventricular pressure (dP/dt) and regional myocardial blood flow were measured during control, occlusion and reperfusion periods. Infarct size was determined by histochemical staining; and myeloperoxidase activity, a marker for tissue neutrophil content, was assessed in normal and infarcted myocardium. The effect of BW755C on the function of isolated neutrophils stimulated with zymosan-activated serum was evaluated by measuring neutrophil degranulation, leukotriene B4 production, superoxide generation and chemotaxis. RESULTS Hemodynamic function and regional myocardial blood flow were similar in control and BW755C-treated animals. BW755C significantly reduced myocardial infarct size compared with that in control animals, as measured by infarct/risk areas by histochemical staining (39 +/- 5% vs. 63 +/- 7%, p < 0.05). Myocardial myeloperoxidase activity was similar in normal, salvaged and infarcted areas in the control and treated groups, indicating that neutrophil accumulation in injured myocardium was unaltered by BW755C. However, this agent attenuated function of isolated, stimulated (zymosan-activated serum) neutrophils. At a concentration of 0.03 mg/ml, BW755C inhibited degranulation (-46%), leukotriene B4 production (-48%) and superoxide generation (-74%), but there was minimal inhibition of chemotaxis in vitro. CONCLUSIONS These findings demonstrate that myocardial infarct size can be reduced by selective inhibition of neutrophil cytotoxic activity without affecting neutrophil migration into injured myocardium.

Effects of dietary supplementation with eicosapentaenoic acid or gamma-linolenic acid on neutrophil phospholipid fatty acid composition and activation responses

Previous data that alimentation with fish oil rich in eicosapentaenoic acid (EPA; 20: 5n-3) or vegetable oil rich in gamma-linolenic acid (GLA; 18: 3n-6) can reduce symptoms of inflammatory skin disorders lead us to determine the effects of dietary supplements of oils rich in EPA or GLA on guinea pig (GP) neutrophil (PMN) membrane potential (δγ), secretion, and Superoxide (O2−) responses. Weanling GPs were initially fed diets supplemented with olive oil (<0.1% EPA; <0.1% GLA) for 2 weeks, followed by a crossover by two sets of animals to diets supplemented with fish oil (19% EPA) or borage oil (25% GLA). At 4-week intervals, 12% sterile casein-elicited peritoneal neutrophils (PMN) were assessed for membrane polyunsaturated fatty acid (PUFA) profiles and FMLP-, LTB4s-, and PMA-stimulated δγ changes, changes in flow cytometrically measured forward scatter (FWD-SC) (shape change), 90‡ scatter (90‡-SC) in cytochalasin B-pretreated-PMN (secretion response), and Superoxide responses. GP incorporated EPA and GLA (as the elongation product, dihomo-GLA or DGLA) into their PMN phospholipids by 4 weeks. The peritoneal PMN of all groups demonstrated broad resting FWD-SC and poor activation-related FWD-SC increases, suggesting in vivo activation. While secretion was comparable in the three groups in response to FMLP, there was a trend toward inhibition of LTB4-stimulated 90‡-SC loss in both fish and borage oil groups. This was significant only with borage oil (21.7±2.1 vs 15.3±1.2% loss of baseline 90‡-SC, olive vs borage;P=0.03). PMN from borage- and fish oil-fed GPs showed a progressively lower O2− response to FMLP than the olive oil group (73.9±3.9 and 42.9±6.8% of olive oil response for borage and fish oils, respectively;P< 0.005 andP<0.01, respectively, at 12 weeks), while PMA-stimulated O2− was inhibited only in the fish oil-fed group and only at 12 weeks (62.0±2.7% of control;P<0.025). We conclude that dietary supplementation with oils rich in PUFAs can modify PMN activation responses.

Patients with adult respiratory distress syndrome (ARDS) demonstrate in vivo neutrophil activation associated with diminished binding of neutrophil-specific monoclonal antibody 31D8.

Neutrophils (PMNs) from patients with adult respiratory distress syndrome (ARDS) were assessed for light scattering, membrane potential, and phagocytic responses using fluorescent probes and flow cytometry to evaluate individual cells. Qualitative and quantitative oxidant responses were measured by nitroblue tetrazolium (NBT) and cytochromec reduction assays, respectively. The results were correlated with the proportion of cells binding the PMN subset-specific monoclonal antibody 31D8. Despite an increased forward scatter signal (4.3±1.6 vs. 1.3±1.1 ARDS vs. control,P=0.041) and spontaneous NBT test (12.6±4.7% vs. 2.5 ±0.8% positive, ARDS vs. control,P=0.033) indicating in vivo priming of ARDS PMNs, there were no significant differences between ARDS and control PMNs in assays of stimulated membrane potential, NBT, and O·2− production or phagocytosis. However, positive correlations between the degree of prestimulus forward light scatter and subsequent O·2− production to FMLP (r=0.673,P=0.006) and between the percentage of bands and the O·2− response to PMA (r=0.660,P =0.003), suggest that the great variability of the ARDS PMN functional responses may relate to varying degrees of in vivo cell priming and/or deactivation. ARDS PMNs demonstrated a significantly lower percentage of 31D8 positive cells (73.4 ±7.5% vs. 94.5±1.6%,P=0.012) and a lower level of 31D8 staining when compared to normals (60.1±10.4% of control level,P=0.001). The lower 31D8 expression did not directly correlate with any functional parameter tested or with the proportion of immature cells. However, patients receiving an intravenous PGE21-infusion demonstrated a significant increase in 31D8 staining relative to controls and inhibition of PMA-stimulated O·2− production. The data suggest that the function of PMNs from ARDS patients varies widely and reflects great in vivo variation in cell priming. While the mechanism responsible for the lowered expression of the 31D8 antigen and its apparent modulation by PGE1 is unknown, 31D8 may be an indirect marker for in vivo stress factors that regulate the preferential release of a structurally distinct PMN subset from the bone marrow.

Shared antigenic determinants between rabbit antihuman brain and rabbit antihuman thymocyte sera: Relationship to the lymphocytotoxic antibodies of systemic lupus erythematosus☆

Abstract Antibodies to human brain, liver, and choroid plexus were produced by immunization of rabbits with fresh autopsy material in Freunds complete adjuvant, and then were absorbed with human AB cells. Rabbit anti-human brain serum was cytotoxic for peripheral blood T cells using the two-stage microcytotoxicity assay. In the presence of complement, it prevented formation of E rosettes and reduced responsiveness to Con A, PHA-P, and allogeneic cells. In the absence of complement, it was strongly mitogenic, much like rabbit anti-human thymocyte sera. Similarly, rabbit anti-human-brain sera could be absorbed with human peripheral blood T cells, but not with B cells or liver. Rabbit anti-human-liver and choroid plexus sera did not exhibit such T-cell specificity. Lymphocytotoxic antibodies were also studied in the cerebrospinal fluid of 11 patients with systemic lupus erythematosus, and of 31 neurologic disease controls. There was no correlation in SLE with presence of CNS disease and either titer or target cell specificity of lymphocytotoxic antibodies. In contrast, patients with multiple sclerosis or meningitis, possessing CSF lymphocytotoxic antibodies, had specificities directed only against T cells, suggesting their origin as autoimmunization with inflamed brain.

Evaluation of prostaglandin E1 for prevention of respiratory failure in high risk trauma patients: A prospective clinical trial and correlation with plasma suppressive factors for neutrophil activation

A group of 48 critically injured patients were entered into a prospective, double-blind, placebo-controlled trial to evaluate the efficacy of early infusion of PGE1 for reducing the incidence of severe respiratory failure and mortality. Secondary assessments examined the effects of the PGE1 infusion on plasma mediated suppression of PMN superoxide production and loss of PMN granule enzyme content. The incidence of severe respiratory failure was lower in the PGE1 group--13% versus 32%, but this did not reach significance. The overall morality was equivalent between the two groups--26% (PGE1) versus 28% (placebo). The suppressive activity of the patient plasma was assayed by measurement of normal PMN superoxide production relative to normal control plasma (ratio P:C). The baseline ratio P:C was 62 +/- 5% in the PGE1 group versus 60 +/- 5% in the placebo group. The day 1 plasma samples showed significant reversal of plasma suppressive activity in the PGE1 group--ratio P:C 88 +/- 5% versus 67 +/- 5% in the placebo group (P less than 0.02). In patients who received the full 7 days of infusion, the plasma suppressive activity remained significantly diminished in the PGE1 group--ratio P:C 77 +/- 4% versus 61 +/- 5% (P less than 0.04). The baseline lysozyme content of patient PMNs relative to that of normal control PMNs (ratio P:C) was 119 +/- 14% in the PGE1 group. A significant loss of lysozyme content was observed in the PGE1 group on day 1 of the infusion--ratio P:C 79 +/- 8% (P less than 0.03), and was associated with a reduction in the plasma suppressive activity.(ABSTRACT TRUNCATED AT 250 WORDS)

T CELL FUNCTION IN CONGENITALLY ASPLENIC (Dh/+) MICE

Publisher Summary This chapter explores the T-cell function in congenitally asplenic (Dh/+) mice. In a study described in the chapter, congenitally asplenic mice were inbred on a B6.CBA background. The mice were maintained at the Experimental Animal Facility at the University of California School of Veterinary Medicine under standard housing conditions; all mice were raised by crossing +/+ females × Dh/+ males. Over a period of approximately 14 months, approximately 100 asplenic and 100 littermate controls were followed unmanipulated. When mice were noted to become moribund, they were immediately sacrificed and autopsied. Sections of brain in addition to all major viscera and identifiable lymph nodes were prepared, stained, and examined. Additionally, groups of 9–12 Dh/+ and +/+ mice were sacrificed at bimonthly intervals, beginning at 4 weeks of age, to determine and compare the thymus weight relative to body weight. The results of this study showed that the spontaneous tumor incidence of hereditarily asplenic mice was approximately the same as littermate controls. The overall morbidity and mortality for asplenic mice was not significantly different than littermates.

Effects of zinc deprivation on heat shock protein (hsp)-65 and-70 gene expression in pregnant rats

Abstract The transcriptional expression of hsp-65 and hsp-70 in the livers of zinc (Zn)-deficient pregnant rats and their fetuses was examined. Despite the induction of severe fetal pathologies, there were no significant differences in either maternal or fetal hsp-65 or hsp-70 expression between Zn-adequate and Zn-deficient groups, although hsp-65 and hsp-70 expression varied in both groups with time of gestation. Thus, while hsp-70 mRNA content is modulated during differentiation and development, the expression of hsp-65 and hsp-70 is not influenced by Zn deprivation, even when it is extensive enough to induce fetal pathology.