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An analysis was conducted of the cytokine profile and inflammatory response in oxazolone sensitized mouse skin. Following exposure to oxazolone, the intralesional production of inflammatory cytokines was demonstrable at the levels of both mRNA and protein. An initial challenge led to a transient increase in tumor necrosis factor-alpha production followed predominately by the T helper (Th)1 cytokine, interferon-gamma. There was a minimal production of interleukin-4, a Th2 cytokine. Continued exposure to oxazolone led to a downregulation of interferon-gamma and an upregulation of interleukin-4 production. A strong relationship was found between interleukin-4 and the inflammatory response, as measured by ear thickness. Similar experiments conducted in mast cell-deficient mice revealed reduced neutrophil influx but only minor changes in cytokine profile. An irritant response induced by chronic exposure of mouse skin to phorbol ester did not reveal any significant interferon-gamma or interleukin-4 response but was characterized by a tumor necrosis factor-alpha response that correlated with the inflammatory response. These observations suggest that the major source of interferon-gamma and interleukin-4 in the oxazolone response may be the infiltrating lymphocytes; whereas the tumor necrosis factor-alpha may result from the local irritation seen with both oxazolone and phorbol ester. At the end of 4 wk of chronic exposure to oxazolone, it was found that serum IgE levels had significantly increased. Histologic analysis of the skin lesion revealed that a mixed infiltrate including eosinophils developed upon repeat exposure to oxazolone. These findings are consistent with an early predominate Th1 response that is reduced and largely replaced with a Th2 response upon chronic T cell activation.

In previous studies, we have used histological methods to characterize cellular changes, and validated the use of the myeloperoxidase (MPO) activity assay to quantitate increased neutrophil infiltration in ischemic stroke. We also identified increased leukotriene B4 (LTB4) binding sites as a potential marker for neutrophil infiltration into focal ischemic tissue. However, these studies were conducted at only one time-point, 24 h after ischemia. In the present study, we examined the full time-course of MPO activity and LTB4 receptor binding following middle cerebral artery occlusion (MCAO) made permanently (PMCAO) or transiently (160 min followed by reperfusion; TMCAO) in spontaneously hypertensive rats, and compared the results to previously characterized histologic changes in these models. Ischemic and contralateral (control) cortical tissue samples were assayed for MPO (U/g wet wt) and [3H]LTB4 receptor binding (fmol/mg protein). Following PMCAO, MPO activity significantly increased as early as 12 h and continued to increase over the next 5 d (p < 0.05). Following TMCAO, MPO activity was significantly elevated already after only 6 h of reperfusion and also continued to increase over the next 5 d of reperfusion (p < 0.05). LTB4 receptor binding and MPO activity were highly correlated during periods when both measures increased together (i.e., 0.5-5 d; p <0.01). However, by 15 d post-MCAO, LTB4 receptor binding remained elevated after MPO activity levels had returned to normal. This persistent LTB4 binding was associated with the significant gliosis that was characterized previously to persist in these models. The time-course of increased MPO activity and initially increased LTB4 binding post-MCAO correspond very well to our previous histological data that characterized the time-course for leukocyte infiltration under these conditions. Therefore, the increased MPO activity over time was associated with initial neutrophil and later mononuclear cell infiltration into ischemic tissue in these models. In addition, the present studies utilized histochemical analysis to demonstrate peroxidase activity in macrophages within the cerebral infarct following MCAO, thus validating that MPO activity originates from the later infiltrating mononuclear cells in addition to the early infiltrating neutrophils that had been previously characterized in the same manner. TMCAO produces a significantly larger and earlier increase in ischemic cortex MPO activity and a similar later increase in MPO activity compared to PMCAO treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

A series of N-hydroxyurea derivatives have been prepared and examined as inhibitors of 5-lipoxygenase. Oral activity was established by examining the inhibition of LTB4 biosynthesis in an ex vivo assay in the mouse. The pharmacodynamic performance in the mouse of selected compounds was assessed using an ex vivo LTB4 assay and an adoptive peritoneal anaphylaxis assay at extended pretreat times. Compounds with an extended duration of action were re-examined as the individual enantiomers in the ex vivo assay, and the (S) enantiomer of N-hydroxy-N-[2,3-dihydro-6-(phenylmethoxy)-3-benzofuranyl]urea, (+)-1a (SB 202235), was selected as the compound with the best overall profile. Higher plasma concentrations and longer plasma half-lives were found for (+)-1a relative to its enantiomer in the mouse, monkey, and dog. In vitro metabolic studies in mouse liver microsomes established enantiospecific glucuronidation as a likely mechanism for the observed differences between the enantiomers of 1a. Enantioselective glucuronidation favoring (-)-1a was also found in human liver microsomes.