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These studies examined the signal transduction mechanisms by which prostaglandin (PG) E2 production can occur in human amnionic WISH cells in response to the stimuli okadaic acid, interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, phorbol-12-myristate-13-acetate (PMA) or combinations of PMA with IL-1beta or TNF-alpha. We also investigated whether WISH cells are capable of producing TNF-alpha or IL-1beta in response to stimulation, because these cytokines can be produced in an autocrine fashion to perpetuate an inflammatory response. Our data indicate that the magnitude of PGE2 production induced by a given stimulus correlated temporally with the level of PGH synthase-2 (PGHS-2) protein. PMA or IL-1beta induced PGE2 production 2 to 4 hr after treatment, whereas the combination of these agents produced the most rapid induction 2 hr after treatment. Only okadaic acid induced the production of both PGE2 and TNF-alpha, after a lag of 12 to 18 hr. PGE2 production by all stimuli was inhibited by dexamethasone, the IL-1 receptor antagonist (IL-1ra), the specific PGHS-2 inhibitor NS-398 and the protein kinase inhibitor staurosporin. In contrast, TNF-alpha production in response to okadaic acid was inhibited by the TNF-converting enzyme inhibitor GI 129471 and staurosporin but was unaffected by either IL-1ra, dexamethasone or NS-398. We conclude that WISH cells are capable of producing bioactive proinflammatory mediators such as TNF-alpha and PGE2 through separable intracellular signal transduction mechanisms. The ability of IL-1ra to reduce PGE2 production caused by all stimuli used suggests an autocrine role for IL-1 in PGHS-2 induction in these cells.

RAW 264.7 macrophages respond to lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) by producing large amounts of nitric oxide (NO) and prostaglandin E2 (PGE2), with maximal production 18-24 h after treatment. Following stimulation with the calcium inophore A23187, cultures of RAW cells also produce modest amounts of leukotrienes. However, the capacity of these cells to produce leukotrienes is transient, beginning 2 h after vehicle or LPS/IFN-gamma treatment, peaking by 4-6 h and absent by 8 h. A-79175, (R(+) N-[3-[5-(4-Fluorophenoxy)-2-furanyl]-1-methyl-2-propynyl]-N-hydroxyurea) a specific inhibitor of 5-lipoxygenase (5-LO), abolished leukotriene production by RAW cells in a dose-dependent, non-cytotoxic fashion while having no effect on PGE2 or NO production. By contrast, nordihydroguaiaretic acid (NDGA) inhibited production of leukotrienes, PGE2 and NO only at doses that were cytotoxic to the RAW cells. Exogenous leukotriene B4 (LTB4) had no effect on either NO or PGE2 production. An inhibitor of NO production, L-N-5-(1-iminoethyl) ornithine HCl (NIO) also did not affect leukotriene or PGE2 production, while dexamethasone blocked PGE2 and NO production, but did not affect leukotriene production in these cells. Taken collectively, these results indicate that there is no interaction between the pathways for leukotriene and nitric oxide production in RAW 264.7 macrophages.

A soluble DNA polymerase was purified 8,000-fold from hepatitis B surface antigen positive serum. The molecular weight of the enzyme by gel filtration was about 1.60 X 10(5), the sedimentation coefficient was 5.5S, the apparent Km for dTTP was 4 micrometer, the optimum pH in the presence of Mg2+ was 9.2, and the pl was 4.7. The enzyme was found in HBsAg-positive sera and required an external primer for activity. The properties of the DNA polymerase were different from hepatitis B virus particle enzyme and from vertebrate and bacterial DNA polymerases. The prevalence of this enzyme did not correlate with HBeAg or particle DNA polymerase in HBsAg-positive sera.

Hematoporphyrin (HP), at concentrations as low as 0.5 microgram/ml, was found to inhibit the in vitro replication of influenza A and herpes simplex viruses, but not of several other viruses. The effect required exposure of the viruses or cells to visible light and was demonstrable when HP was administered shortly before virus inoculation or during the infection. In studies on the mechanism of action of HP, we found that in the presence of light, HP caused decomposition of GMP but not of various other nucleosides. It caused breakdown of yeast tRNA and inhibited polymerization of RNA and DNA by influenza virus and HSV-1-specific polymerases as well as some other polymerases isolated from bacterial and mammalian sources. Protective effects of HP and light were demonstrable in embryonated eggs infected with the WSN and PR8 strains of influenza A virus and in mice infected with the WSN strain. HSV-1-induced keratitis in rabbits and HSV-2-induced dermatitis in mice were not responsive to HP treatment.