Standardization and comparison of an XTT-based TNF-alpha bioassay with a TNF-alpha ELISA.

Dept. of Biology, University of Wisconsin-Eau Claire 54702, USA.
BioTechniques (Impact Factor: 2.95). 03/1998; 24(2):232-4, 236-8.
Source: PubMed
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    ABSTRACT: Cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha can play pathogenetic or protective roles in stroke. They are increased in the brain after experimental ischemia and in the CSF of patients with stroke. However, their presence in the periphery is still controversial. To determine the source and time-course of cytokines in blood of stroke patients, IL-6 and TNF-alpha release from blood cells and serum levels were determined in 40 patients on days 1 through 2, 4, 10, 30, and 90 after stroke. Twenty healthy age-matched volunteers were used as controls. IL-6 and TNF-alpha release from stimulated blood cells was increased in stroke patients, compared to controls. A peak response (+224%) was observed at day 4 for IL-6, while TNF-alpha release was largely and significantly increased (about three-fold compared to controls) from day 1 to 2 until day 90 after stroke. The increase in IL-6 release was significantly higher in ischemic, compared to hemorrhagic strokes, at days 1 and 4. Circulating IL-6 was increased at each time point. The ischemic processes in the CNS induces a long-lasting activation of IL-6 and TNF-alpha production in peripheral blood cells, which are a major source of serum cytokines after stroke.
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    ABSTRACT: Macrophage-induced lung inflammation contributes substantially to respiratory failure during Pneumocystis carinii pneumonia. We isolated a P. carinii cell wall fraction rich in glucan carbohydrate, which potently induces TNF-alpha and macrophage-inflammatory protein-2 generation from alveolar macrophages. Instillation of this purified P. carinii carbohydrate cell wall fraction into healthy rodents is accompanied by substantial increases in whole lung TNF-alpha generation and is associated with neutrophilic infiltration of the lungs. Digestion of the P. carinii cell wall isolate with zymolyase, a preparation containing predominantly beta-1,3 glucanase, substantially reduces the ability of this P. carinii cell wall fraction to activate alveolar macrophages, thus suggesting that beta-glucan components of the P. carinii cell wall largely mediate TNF-alpha release. Furthermore, the soluble carbohydrate beta-glucan receptor antagonists laminariheptaose and laminarin also substantially reduce the ability of the P. carinii cell wall isolate to stimulate macrophage-inflammatory activation. In contrast, soluble alpha-mannan, a preparation that antagonizes macrophage mannose receptors, had minimal effect on TNF-alpha release induced by the P. carinii cell wall fraction. P. carinii beta-glucan-induced TNF-alpha release from alveolar macrophages was also inhibited by both dexamethasone and pentoxifylline, two pharmacological agents with potential activity in controlling P. carinii-induced lung inflammation. These data demonstrate that P. carinii beta-glucan cell wall components can directly stimulate alveolar macrophages to release proinflammatory cytokines mainly through interaction with cognate beta-glucan receptors on the phagocyte.
    The Journal of Immunology 05/2000; 164(7):3755-63. DOI:10.4049/jimmunol.164.7.3755 · 4.92 Impact Factor