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A series of 3-substituted 2-piperidinone (delta-valerolactam) and hexahydro-2H-azepin-2-one (epsilon-caprolactam) derivatives were prepared and evaluated as anticonvulsants in mice. In the 2-piperidinone series, 3,3-diethyl compound 7b is the most effective anticonvulsant against pentylenetetrazole-induced seizures (ED50, 37 mg/kg; PI (TD50/ED50), 4.46), and 3-benzyl compound 4c (ED50, 41 mg/kg; PI, 7.05) is the most effective anticonvulsant against seizures induced by maximal electroshock. By contrast, none of the epsilon-caprolactams tested had anticonvulsant effects below doses causing rotorod toxicity. log P values were correlated with neurotoxicity and [35S]TBPS displacement, but not with anticonvulsant activity. Electrophysiological evaluations of selected compounds from each series indicated that both the delta-valero-lactams and epsilon-caprolactams potentiated GABA-mediated chloride currents in rat hippocampal neurons.

Polymorphonuclear leukocytes (PMN) from human colostrum were compared with blood and oral exudate PMN from the same donors for their locomotive, respiratory burst, phagocytic, and shape change (polarization) capabilities. Blood PMN were functionally superior to PMN from other sites. Colostrum PMN were similar to oral exudate PMN in all areas except locomotive responses. Exposure of blood PMN to aqueous human colostrum resulted in decreased stimulated adherence to plastic, decreased bactericidal activity against Staphylococcus aureus, reversible induction of cellular shape change, and reversible decreases in cellular deformability. The colostrum effects on PMN shape change and deformability were accompanied by significant increases in cytoskeleton-associated actin. PMN isolated from colostrum have suppressed functions, consistent with their being exudate cells. In addition, the colostrum environment effectively suppresses multiple functions in PMN from blood, these effects being mediated in part by rapid cytoskeletal assembly. PMN in colostrum do not appear to be beneficial to the breast-fed infant due to deficiencies in function.

During a phase I trial of the genetically engineered hematopoietic growth factor PIXY321 (granulocyte-macrophage colony-stimulating factor/interleukin-3 [IL-3] fusion protein), we examined the effects of PIXY321 treatment on human polymorphonuclear leukocyte (PMN) locomotive, respiratory burst, and phagocytic responses. PIXY321 treatment was associated with transient suppression of both unstimulated locomotion and chemotaxis responses to multiple stimuli, as well as significant transient enhancement of formyl peptide-stimulated H2O2 production. No effects on opsonic phagocytosis of Staphylococcus aureus were observed. In vitro exposure of control PMN to PIXY321 resulted in suppression of unstimulated locomotion/chemotaxis and enhancement of formyl peptide-stimulated H2O2 production but had no effects on phagocytosis. When patient cells were exposed in vitro to PIXY321 during treatment, suppression of chemotaxis and enhancement of H2O2 production were observed before PIXY321 treatment, but these effects diminished during treatment. The in vivo and in vitro exposure effects of PIXY321 treatment on PMN function are similar to those of the parent molecule, granulocyte-macrophage colony-stimulating factor (GM-CSF).

During a phase I trial of interleukin-1 alpha (IL-1 alpha) in patients with ovarian carcinomas, the effects of this treatment on blood granulocyte respiratory burst and locomotive responses were examined. Differences in baseline granulocyte function in patients as well as dose-related effects of IL-1 alpha treatment were observed. Patients enrolled early in the trial (low-dose patients) had significantly lower locomotive responses before treatment than their paired controls; these low responses normalized after 5 days of continuous-infusion IL-1 alpha treatment. Patients enrolled later (high-dose patients) had normal locomotive responses before treatment and IL-1 alpha treatment was associated with suppression of responses to selected stimuli at the end of treatment. Pretreatment respiratory burst responses in both low- and high-dose patient groups were essentially normal, but the rates of granulocyte H2O2 production following phorbol myristate acetate stimulation became significantly less than control values at the end of treatment. In vitro exposure of either patient or control cells to 150 U/ml IL-1 alpha did not alter their locomotive or respiratory burst responses, suggesting the observed in vivo effects were not mediated directly by IL-1 alpha. Treatment with IL-1 alpha is associated with changes in ex vivo granulocyte function that are related to the IL-1 alpha dose. Treatment with low doses of IL-1 alpha may provide a means of normalizing abnormal polymorphonuclear leukocyte function in some patients with ovarian malignancies.

Human colostrum contains several antioxidants which prevent the detection of human polymorphonuclear leukocyte (PMN) respiratory burst products. Using column chromatography to fractionate colostrum, two peaks of antioxidant activity were resolved away from colostral proteins and further characterized. One peak contained both cytochrome c-reducing activity and H2O2-depleting activity. This peak had the chromatographic, spectral, and antioxidant characteristics of ascorbate, and by high performance liquid chromatography (HPLC) methods, was shown to contain ascorbate as well as at least four other materials. The antioxidant activity in this peak was totally ascorbate oxidase sensitive and partially uricase sensitive. The other peak contained only H2O2-depleting activity and had the chromatographic, spectral, and antioxidant characteristics of uric acid. By HPLC, uric acid was the only component in this peak and its antioxidant activity was completely uricase sensitive and ascorbate oxidase resistant. Colostral uric acid levels were measured in eight postpartum women and found to be approximately one-third of simultaneously determined serum uric acid levels. Colostrum contains at least two separate antioxidants, one of which is ascorbate-like and the other is uric acid. We speculate that these antioxidants may function in human colostrum as traps for neutrophil-generated reactive oxygen metabolites.

A murine monoclonal IgM antibody, M3, which interferes with both polymorphonuclear leukocyte (PMN) phagocytosis and bactericidal activity, was used to examine the subcellular location of antigens bearing 3-fucosyllactosamine (CD15 antigens) within this cell type. Percoll gradient-separated secondary granule fractions were rich in CD15 antigens, with at least seven antigens recognizable in SDS-PAGE/electroblot studies. Sonication/sedimentation experiments using secondary granule fractions showed that both soluble and sedimentable CD15 antigens were present. Exposure of purified PMN to the secondary granule secretagogue phorbol myristate acetate caused extracellular release of two or three CD15 antigens, which could be purified by immunoprecipitation using antibody M3. Triton X-114 phase-partition experiments showed that secondary granule fraction CD15 antigens could be partitioned into hydrophilic (aqueous phase) and hydrophobic (detergent phase) antigens, suggesting that several of these antigens were integral secondary granule membrane components. Ultrastructurally, PMN intracellular granules showed two patterns of CD15 expression, localization over both granule matrix/granule membrane and localization to only granule membrane. Colocalization studies showed that lactoferrin and CD15 antigens were both present in a subset of intracellular granules, confirming a secondary granule location for these antigens.

Locomotion and oxidative metabolism of polymorphonuclear leukocytes from 15 patients receiving recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) were examined in vitro. At the end of each GM-CSF treatment course, polymorphonuclear leukocyte (PMN) chemotactic responses were suppressed and no enhancement of formyl-peptide-stimulated superoxide production was observed. The priming of PMN superoxide production normally seen after in vitro GM-CSF exposure was also blunted in these cells. By using control donor PMN, two membrane-fluidizing agents, pentoxifylline and butanol, were shown to normalize suppressed PMN chemotaxis caused by in vitro GM-CSF (1 nM) exposure. Pentoxifylline, but not butanol, also reversed the effects of in vitro GM-CSF on PMN superoxide production. When PMN obtained from six patients at the end of GM-CSF therapy were exposed to pentoxifylline in vitro, the chemotactic suppression typically observed was significantly improved. The data suggest that GM-CSF may affect PMN function via mechanisms involving membrane fluidity or cell deformability or both.

Because it has recently been hypothesized that human milk is antiinflammatory, the effects of aqueous human colostrum on human polymorphonuclear leukocyte (PMN) respiratory burst activity and selected enzymatic activities was examined. Aqueous colostrum was found to spontaneously reduce ferricytochrome C in a concentration-dependent manner, prohibiting use of the standard assay to measure superoxide production. It also caused a significant concentration-dependent prolongation of the lagtime from stimulation of PMN with phorbol myristate acetate to the appearance of hydrogen peroxide. Substitution of an enzymatic peroxide-generating system for PMN did not alter the effect of colostrum. Colostrum also suppressed myeloperoxidase activity and lysozyme activity, but not beta-glucuronidase activity in PMN lysates. Inclusion of colostrum in an in vitro assay of PMN-mediated cell detachment significantly suppressed this PMN-mediated effect. These data demonstrate that aqueous human colostrum significantly interferes with PMN oxygen metabolic and enzymatic activities that are important in the mediation of acute inflammation.

Human colostrum manifests antioxidant properties, being capable of spontaneous reduction of cytochrome c, depletion of polymorphonuclear leukocyte-produced H2O2 and protection of epithelial cells from PMN-mediated detachment. These activities can be electrophoretically concentrated at either 3.5 kD or 50 kD dialysis membranes at mildly alkaline pH. They are progressively lost under increasingly alkaline conditions. They are resistant to 1-mM N-ethylmaleimide. Examination of a series of antioxidant compounds showed that ascorbate manifests several characteristics of colostrum, being able to reduce cytochrome c and deplete H2O2 but not altering PMN-mediated HEp2 cell detachment. Addition of ascorbate oxidase to colostrum decreased its cytochrome c-reducing activity by more than 85%, decreased its H2O2-depleting activity by nearly 50%, but did not alter its ability to protect HEp2 cells, all suggesting heterogeneity of colostral antioxidant activities. Treatment of colostrum with an enzymatic system (xanthine + xanthine oxidase) known to destroy ascorbate's cytochrome c-reducing activity yielded paradoxical results, decreasing colostral cytochrome c reduction in a dose-related manner, while increasing its H2O2-depleting activity. These studies demonstrate that a colostral component similar to ascorbate, a known antioxidant compound is responsible for the majority of colostral cytochrome c-reducing activity, for about half of its H2O2-depleting activity, and little, if any, of its protective effect on HEp2 cells. Thus colostral antioxidant activity is heterogeneous.