Arachidonic Acid Metabolism In Murine Fibrosarcoma Cells With Differing In Vivo And In Vitro Characteristics

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/144314/1/ijc1985363383.pd

Regulation of cytokine gene expression by reactive oxygen and reactive nitrogen intermediates

Reactive oxygen intermediates (ROI), reactive nitrogen intermediates (RNI), and cytokines are frequent companions at sites of acute inflammation. Previous work has established a clear link between the production of cytokines and the subsequent generation of ROI and RNI. However, more recent data indicates that ROI and RNI not only serve as end‐stage effector molecules of pathogen destruction and tissue injury, but also as initiators of acute inflammation. Specifically, ROI and RNI will up‐regulate cytokine gene expression since antioxidants inhibit interleukin 8 (IL‐8) production and do not decrease production of other cytokines. Treatment with hydroxyl radical scavengers such as dimethyl sulfoxide (DMSO) will decrease the production of IL‐8 in stimulated human whole blood, fibroblasts, type II epithelial cells, and hepatoma cells, but not other cytokines. Addition of exogenous ROI will increase IL‐8 production in these same cells. Inhibition of nitric oxide synthase will decrease production of IL‐8, whereas addition of nitric oxide (NO)‐generating compounds will increase production of IL‐8. The hydroxyl radical appears to be the final common pathway of cell activation for IL‐8 synthesis, since DMSO will inhibit the NO‐driven production of IL‐8. Our data indicate that ROI and RNI can serve as intracellular second messengers to induce IL‐8 gene expression.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141605/1/jlb0471.pd

Chemiluminescence of asbestos-activated macrophages

Chemiluminescence, a measure of reactive oxygen release by phagocytes, was compared in peritoneal exudate macrophages elicited with chrysotile asbestos, Corynebacterium parvum and saline. Chrysotile asbestos- and C. parvum-activated macrophages produced significantly more chemiluminescence than saline-elicited macrophages. In a second series of experiments the ability of opsonized chrysotile asbestos to act as a trigger for the release of chemiluminescence was tested. Opsonized chrysotile asbestos produced a dose-related release of chemiluminescence from activated macrophages except at the highest dose where chemiluminescence was reduced due, possibly, to a toxic effect of chrysotile during the assay. Opsonized latex also triggered a dose-related chemiluminescent response from activated macrophages. The potential role of toxic reactive oxygen species, released from macrophages, in the development of asbestos-related pulmonary inflammation and fibrosis are discussed

3‐Deaza‐Adenosine Inhibition of Stimulus‐Response Coupling in Human Polymorphonuclear Leukocytes

In an effort to define better the functional role of S‐adenosyl‐methionine‐mediated methylation reactions in modulating polymorphonuclear (PMN) functional responses to chemotactic stimuli, we investigated the effects of 3‐deaza‐adenosine (3‐DZA), a known inhibitor of methylation reactions in phagocytic cells, on formyl methionyl‐leucyl‐phenylalanine (FMLP)‐induced responses in human PMN leukocytes. Using the fluorescent cyanine dye 3,3’‐dipropylthiocarbocyanine (di‐S‐C3‐(5)) as an optical probe of membrane potential we observed that 3‐DZA at concentrations that inhibit FMLP‐induced O2− production does not significantly alter FMLP‐induced changes in transmembrane potential. Additional studies showed an inhibitory effect of 3‐DZA on FMLP‐induced PMN pinocytosis and to a lesser degree on FMLP‐induced degranulation. However, pretreatment of PMNs with 3‐DZA did not alter FMLP‐induced changes in Quin‐2 fluorescence, an indicator of changes in intracellular calcium levels. These findings demonstrate a dissociation between chemotactic factor‐induced cell membrane depolarization, changes in intracellular calcium, and specific neutrophil functional responses and suggest that chemotactic factor‐induced changes in transmembrane potential and intracellular calcium are independent of chemotactic factor‐induced methylation reactions. Furthermore, 3‐DZA did not alter phorbol myristate acetate induced O2− production or fluid pinocytosis indicating a stimulus specificity for the inhibitory effects of this agent on O2− production.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141044/1/jlb0121.pd

A simple method for the purification of human peripheral blood monocytes : A substitute for Sepracell-MN

Sepracell-MN has provided a simple, rapid means of isolating peripheral blood monocytes. However this product is no longer available. Consequentially we have developed a Percoll gradient wich matches Sepracell-MN in simplicity and yield of monocytes. Using this Percoll gradient, an average of 7 x 106 monocytes with a purity of 83% were obtained from 30-40 ml of blood. These monocytes were at least 97% viable and responded to chemotactic stimuli in comparable numbers to those prepared using Sepracell-MN.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29026/1/0000056.pd

Iron released from transferrin at acidic pH can catalyse the oxidation of low density lipoprotein

AbstractLow density lipoprotein (LDL) oxidation within the arterial wall may contribute to the disease of atherosclerosis. We have investigated the conditions under which transferrin (the major iron-carrying protein in plasma) may release iron ions to catalyse the oxidation of LDL. Transferrin that had been incubated at pH 5.5 released approximately 10% of its bound iron in 24 h, as measured by ultrafiltration and atomic absorption spectroscopy. Furthermore, transferrin co-incubated with LDL and l-cysteine at pH 5.5 resulted in the oxidation of the LDL as measured by thiobarbituric acid-reactive substances and electrophoretic mobility. This effect was observed at transferrin concentrations as low as 40% of its average plasma concentration. The release of iron from transferrin in atherosclerotic lesions due to a localised acidic pH may help to explain why LDL oxidation occurs in these lesions

Oxidative stress: acute and progressive lung injury

Oxidative stress in lung often occurs in humans during acute lung injury (ALI) and in the acute respiratory distress syndrome. The lung inflammatory response may proceed to the development of pulmonary fibrosis, a devastating complication that occurs in premature infants after prolonged exposure to high oxygen concentrations. Oxidant-related ALI can be induced by airway deposition of lipopolysaccharide or IgG immune complexes, resulting in activation of recruited neutrophils and residential macrophages, whose oxidants and proteases produce reversible ALI. In the presence of a powerful trigger of leukocytes (phorbol myristate acetate), or following intrapulmonary deposition of enzymes that generate oxidants, extensive endothelial and epithelial damage and destruction occurs, overwhelming repair mechanisms of lung and resulting in pulmonary fibrosis. How residential or circulating stem cells participate in regeneration of damaged/destroyed cells may provide clues regarding therapy in humans who are experiencing lung inflammatory damage.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79133/1/j.1749-6632.2010.05552.x.pd

Reprogramming of Human Peripheral Blood Cells to Induced Pluripotent Stem Cells

Embryonic stem cells are pluripotent cells derived from the inner cell mass of the developing embryo that have the capacity to differentiate into every cell type of the adult (Evans and Kaufman, 1981, Martin, 1981, Martin and Evans, 1975 and Thomson et al., 1998). The generation of patient-specific pluripotent cells is therefore an important goal of regenerative medicine. A major step to achieve this was the recent discovery that ectopic expression of defined transcription factors induces pluripotency in somatic cells (Lowry et al., 2008, Park et al., 2008b, Takahashi et al., 2007 and Yu et al., 2007). Until now, the most common source from which to derive human iPSCs has been skin fibroblasts (Lowry et al., 2008, Park et al., 2008a, Park et al., 2008b, Takahashi et al., 2007 and Yu et al., 2009). However, the requirement for skin biopsies and the need to expand fibroblast cells for several passages in vitro represent a hurdle that must be overcome to make iPSC technology broadly applicable. Peripheral blood can be utilized as an easily accessible source of patient tissue for reprogramming. Here we derived iPSCs from frozen human peripheral blood samples. Some of the iPSCs had rearrangements of the T cell receptor (TCR), indicating that T cells can be reprogrammed to pluripotency.National Institutes of Health (U.S.) (Grant 5-RO1-HDO45022)National Institutes of Health (U.S.) (Grant 5-R37-CA084198)National Institutes of Health (U.S.). (Grant 5-RO1-CA087869)National Center for Research Resources (U.S.) (Grant UL1 RR025758

Monocyte urokinase expression: modulation by interleukins

This study delineates the regulatory effect of interleukin‐1 (IL‐1) and interleukin‐2 (IL‐2) on monocyte plasminogen activator (PA) activity. Mononuclear phagocytes regulate net PA activity by modulating the expression of urokinase‐type PA (uPA) and a specific plasminogen activator inhibitor, PA1‐2. To understand the regulation of mononuclear phagocyte PA activity, it is important to compare the expression of uPA and PAI‐2. In this study, we determined the relative abundance of secreted PA and PA inhibitor activity in human monocyte‐conditioned medium after stimulation with human recombinant IL‐1 or IL‐2. In agreement with our previous description of tumor necrosis factor‐α and interferon‐ γ stimulation of mononuclear phagocytes, we found no detectable PA activity in conditioned medium. Both IL‐1 and IL‐2 had dose‐dependent effects, significantly up‐ regulating PA inhibitor activity in monocyte‐conditioned medium (up to 11‐fold). To further investigate the mechanism underlying this effect, Northern blot analysis was done to measure steady‐state mRNA for uPA and PAI‐2. Consistent with the increase in secreted PA inhibitor activity, we found that both IL‐1 and IL‐2 significantly increased steady‐state mRNA for PAI‐2. In addition, however, both IL‐1 and IL‐2 increased steady‐state mRNA for uPA. IL‐1 appears to increase mRNA for uPA to a greater extent than does IL‐2. We conclude that IL‐1 and IL‐2 modulate monocyte proteolytic activity by increasing expression of uPA and PAI‐2 with a resultant predominance of PAI‐2. We further conclude that cytokine‐specific regulation of plasminogen activity is achieved partly by varying the proportionate expression of uPA and PAI‐2.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141561/1/jlb0598.pd