We investigated prostanoid biogenesis in human colonic fibroblasts (CCD-18Co and 5 primary fibroblast cultures) and epithelial cell lines (NCM460, T84, HT-29, and LS 174T) and the effect of PGE2 on fibroblast morphology. Cytokine-stimulated PGE2production was measured. PGH synthase-1 and -2 (PGHS-1 and -2) protein and mRNA expression were evaluated. Basal PGE2 levels were low in all cell types (0.15–6.47 ng/mg protein). Treatment for 24 h with interleukin-1β (IL-1β; 10 ng/ml) or tumor necrosis factor-α (50 ng/ml), respectively, elicited maximal 25- and 6-fold inductions of PGE2 synthesis in CCD-18Co cultures and similar results in primary fibroblast cultures; maximal inductions with IL-1β in colonic epithelial cell lines were from zero to fivefold. Treatment of CCD-18Co fibroblasts with IL-1β caused maximal 21- and 53-fold increases, respectively, in PGHS-2 protein and mRNA levels without altering PGHS-1 expression. PGE2 (0.1 μmol/l) elicited a dramatic shape change in selected fibroblasts. Colonic fibroblasts are potentially important as cytokine targets and a source of and target for colonic prostanoids in vivo.

Endotoxins, the lipopolysaccharide (LPS) moieties on the bacterial cell wall, cause many of the pathological features of Gram-negative septicemia. Tumor necrosis factor (TNF), primarily a product of monocyte/macrophages, has been shown to mediate many of the pathophysiological effects of endotoxin. Kupffer cells, the largest macrophage population in the body, release TNF when stimulated by LPS in vitro. A recombinant human hybrid interferon-alpha A/D (rIFN-alpha) markedly inhibited this LPS-elicited TNF production by Kupffer cells. The effects of rIFN-alpha were further tested in C57BL/6 mice receiving a lethal dose (400 micrograms/mouse) of LPS. All LPS-treated mice died within 2 days. Pretreatment with rIFN-alpha 1 h before LPS challenge improved the survival at 3 days to 22% (5/23, p < 0.04). In contrast, rIFN-alpha was more effective when administered 20 min after LPS injection, increasing the survival rate to 81% (13/16, p < 0.0001). TNF mRNA expression in the liver and spleen 50 min after LPS challenge, and plasma TNF 1.5 h after LPS were also reduced by either pretreatment or post-treatment with rIFN-alpha. Subsequently, experiments were carried out to test the efficacy of delayed rIFN-alpha treatment. A significant protective effect was still apparent when rIFN-alpha was administered 6, 10 and even 14 h (81%, 62% and 28% survival, respectively) after LPS challenge when serum TNF levels had already returned to near baseline. These experimental results suggest that rIFN-alpha might have a therapeutic potential for the prevention and treatment of the deleterious effects associated with endotoxemia besides mechanisms initially blocking TNF production.

n-Butyrate, a short-chain aliphatic carboxylic acid with pleiotropic actions, is present at high concentrations in the portal circulation and thus may play an important role in the regulation of specific gene expression in the mammalian liver. We report here that n-butyrate can increase substantially the level of plasminogen activator inhibitor type 1 (PAI-1) messenger RNA (mRNA) in Hep G2 cells, up to eightfold above control cultures. Maximal effects occurred at a concentration of 3 mmol/L n-butyrate and with a treatment period of 8 to 12 hours. Increases in PAI-1 mRNA were accompanied by modest increases (twofold) in the encoded protein as assessed by specific enzyme-linked immunosorbent assay and by [35S]methionine incorporation into immunoprecipitable PAI-1 in the culture medium. Nuclear run-on studies showed that the rate of transcription of the PAI-1 gene did not appear altered by treatment with 3 mmol/L n-butyrate for 6 hours. The increases in steady-state PAI-1 mRNA caused by exposure to n-butyrate can be blocked by cycloheximide. Enhanced stability of mature PAI-1 transcript could not be demonstrated in Hep G2 cells treated with the carboxylic acid. We have reported previously that n-butyrate can reduce the level of beta-galactoside alpha 2,6-sialyltransferase expression in Hep G2 cells. That effect was attenuated with inhibitors of protein and RNA synthesis and was mediated at the post-transcriptional level. Thus, n-butyrate can influence the expression of multiple genes in this hepatoblastoma cell through its actions on events that appear to be posttranscriptional. These observations may be relevant to the normal physiology of the mammalian liver because of the high concentrations of n-butyrate and related compounds to which the organ is ordinarily exposed.

n-Butyrate, a short-chain aliphatic carboxylic acid n-Butyrate, a four-carbon short-chain aliphatic carwith pleiotropic actions, is present at high concentra-boxylic acid, and compounds structurally related to it tions in the portal circulation and thus may play an im-exert numerous actions on cells in culture. Regulation portant role in the regulation of specific gene expression of cellular differentiation, proliferation, receptor abunin the mammalian liver. We report here that n-butyrate dance, specific gene expression, and the activities of can increase substantially the level of plasminogen acti-several key enzymes are among the pleiotropic effects vator inhibitor type 1 (PAI-1) messenger RNA (mRNA) observed with n-butyrate treatment.1-8 Although thesein Hep G2 cells, up to eightfold above control cultures.activities are well documented in vitro, whether theyMaximal effects occurred at a concentration of 3 mmol/L occur in the whole body remains uncertain. Human n-butyrate and with a treatment period of 8 to 12 hours.tissues exposed to extremely high (mmol/L range) conIncreases in PAI-1 mRNA were accompanied by modest increases (twofold) in the encoded protein as assessed centrations of short-chain fatty acids (SCFA) include by specific enzyme-linked immunosorbent assay and by the colon and liver. 9,10 Cells derived from these tissues complex with tissue and urinary forms of plasminogen activator. 13,14 The polypeptide is released from the cell of origin and deposited in the extracellular matrix, Abbreviations: SCFA, short-chain fatty acids; mRNA, messenger RNA; PAIwhere it functions as a key modulator of the pericellu-1, plasminogen activator inhibitor type 1; cDNA, complementary DNA.From the

A 70-year-old man was found to have at least 12 type I gastric carcinoids and microcarcinoidosis. We performed an extended octreotide suppression test to determine if the tumors were gastrin-dependent and would likely regress after antrectomy. He was given an octreotide infusion at 12.5-25 mcg/h for 86 hr followed by depot octreotide 20 mg intramuscularly every four weeks for eight months. Fasting serum gastrin and chromogranin A levels were measured, and endoscopy with biopsies was performed before and after the infusion and at five months and eight months. Total RNA was extracted for quantitation of histidine decarboxylase mRNA using real-time PCR. Fasting serum gastrin decreased from 306 pg/ml pretreatment to 31 pg/ml at the end of infusion and 115 pg/ml at eight months. Chromogranin A decreased from four to six times the upper limit of normal to normal. Tissue histidine decarboxylase mRNA decreased 50-fold. At eight months, only a few diminutive nodules were present on endoscopy. These results demonstrated that the carcinoid tumors in this patient were under neuroendocrine control and were expected to respond to antrectomy.