A pathway for entry of reoviruses into the host through M cells of the respiratory tract.

Many microorganisms gain access to the systemic circulation after entering the respiratory tract. The precise pathways used to cross the mucosal barriers of the lungs have not been clearly described. We have used the mammalian reoviruses in order to determine the pathway that a systemic virus uses to penetrate the mucosal barrier and enter the systemic circulation after entering the airways of the lungs. Reoviruses enter through pulmonary M cells, which overlie bronchus-associated lymphoid tissue, and subsequently spread to regional lymph nodes. Thus, the pathway through M cells represents a strategy by which viruses and probably other microorganisms can penetrate the mucosal surface of the respiratory tract and thereby enter the systemic circulation.

Pharmacological rescue of mutant p53 conformation and function.

Compounds that stabilize the DNA binding domain of p53 in the active conformation were identified. These small synthetic molecules not only promoted the stability of wild-type p53 but also allowed mutant p53 to maintain an active conformation. A prototype compound caused the accumulation of conformationally active p53 in cells with mutant p53, enabling it to activate transcription and to slow tumor growth in mice. With further work aimed at improving potency, this class of compounds may be developed into anticancer drugs of broad utility.

Biochemical effects and therapeutic potential of tunicamycin in murine L1210 leukemia.

Tunicamycin, an antibiotic which specifically inhibits the dolichol-mediated synthesis of glycoproteins, significantly decreased the incorporation of tritiated D-mannose and D-glucosamine into L1210 ascites leukemia cell glycoproteins at concentrations which affected the biosynthesis of proteins minimally. Mice receiving inoculations of L1210 cells pretreated with 10 microM tunicamycin in vitro survived nearly twice as long as did mice receiving implants of untreated tumor cells. A nonlethal dose of X-irradiation (350 rads) to mice 24 hr prior to receiving their inoculation of tunicamycin-treated L1210 cells prevented this increase in life span. Thirty-eight % of the long-term surviving mice which received 1 X 10(5) L1210 cells pretreated with 10 microM tunicamycin in vitro were then resistant to a subsequent challenge with 10(6) untreated L1210 ascites cells. Direct i.p. administration of tunicamycin to mice resulted in potent liver toxicity (50% lethal dose, 2.0 mg/kg) which obviated any therapeutic efficacy when administered to L1210 ascites tumor-bearing mice. The administration of nontoxic levels of D-mannose prior to the administration of tunicamycin decreased the toxicity of the antibiotic in vivo and, when combined with D-mannose in vitro, exhibited cytotoxic additivity in terms of the inhibition of L1210 leukemic cell growth. A therapeutic regimen incorporating a 24-hr infusion of the sugar prior to multiple administrations of tunicamycin gave evidence of a small therapeutic response in terms of the survival of tumor-bearing mice. These results suggest that tunicamycin, an inhibitor of glycoprotein biosynthesis, might be able to alter tumor cell growth and immunogenicity provided that host liver toxicity is diminished.

Inhibition of glycoprotein biosynthesis by the inducers of HL-60 cell differentiation, aclacinomycin A and marcellomycin.

The trisaccharide-containing anthracyclines aclacinomycin A (ACM) and marcellomycin (MCM) have been shown by our laboratory to be potent inducers of HL-60 leukemia cell maturation, while the monosaccharide-containing anthracyclines Adriamycin and pyrromycin were inactive and significantly less potent, respectively, as initiators of the differentiation of these malignant cells. We have now observed that ACM and MCM are potent inhibitors of both total glycoprotein synthesis and the formation of lipid-linked oligosaccharide intermediates in intact HL-60 cells. This inhibitory activity was both concentration and time dependent and was maximal after 12 hr exposure to 30 nM ACM or MCM, conditions under which both cell growth and total protein synthesis were maintained at levels equal to those of untreated cells. In contrast, exposure of HL-60 cells to pyrromycin or Adriamycin, even at cytotoxic concentrations, did not result in specific decreases in the synthesis of glycoproteins containing asparagine-linked oligosaccharides. Maximum inhibition of the formation of lipid-linked intermediates by ACM or MCM preceded the loss of cell surface transferrin-binding activity (maximal at 24 hr), which in turn preceded the minimal exposure time necessary for significant induction of differentiation by either of the active anthracyclines (36 hr). These findings demonstrate a new biochemical site of action for ACM and MCM and suggest that this activity is involved in the induction of terminal differentiation of HL-60 leukemia cells by these antitumor agents.

Induction of apoptosis and cell cycle arrest by CP-358,774, an inhibitor of epidermal growth factor receptor tyrosine kinase.

The epidermal growth factor receptor (EGFR) is overexpressed in a significant percentage of carcinomas and contributes to the malignant phenotype. CP-358,774 is a directly acting inhibitor of human EGFR tyrosine kinase with an IC50 of 2 nM and reduces EGFR autophosphorylation in intact tumor cells with an IC50 of 20 nM. This inhibition is selective for EGFR tyrosine kinase relative to other tyrosine kinases we have examined, both in assays of isolated kinases and whole cells. At doses of 100 mg/kg, CP-358,774 completely prevents EGF-induced autophosphorylation of EGFR in human HN5 tumors growing as xenografts in athymic mice and of the hepatic EGFR of the treated mice. CP-358,774 inhibits the proliferation of DiFi human colon tumor cells at submicromolar concentrations in cell culture and blocks cell cycle progression at the G1 phase. This inhibitor produces a marked accumulation of retinoblastoma protein in its underphosphorylated form and accumulation of p27KIP1 in DiFi cells, which may contribute to the cell cycle block. Inhibition of the EGFR also triggers apoptosis in these cells as determined by formation of DNA fragments and other criteria. These results indicate that CP-358,774 has potential for the treatment of tumors that are dependent on the EGFR pathway for proliferation or survival.

From oncogene to drug: development of small molecule tyrosine kinase inhibitors as anti-tumor and anti-angiogenic agents.

The confluence of two distinct but related activities in the past 10 years has dramatically accelerated efforts towards the discovery and development of novel drugs to treat cancer. The first is a rapidly emerging understanding that a number of distinct tyrosine kinases play roles in diverse but fundamentally important aspects of tumor progression (growth, survival, metastasis and angiogenesis). The second is the discovery that small molecule compounds have the capacity to potently and selectively inhibit the biochemical function of tyrosine kinases by competing for ATP binding at the enzyme catalytic site. These observations have been conjoined in major efforts to bring forward into clinical development novel cancer drugs with the potential to provide both clinical efficacy and improved tolerability. The focus of this review is on the development of small molecule tyrosine kinase inhibitors, and does not extend to other approaches that could be applied to disrupt the same pathways in clinical tumors (receptor and/or ligand-competitive antibodies, intrabodies, antisense ribonucleotides, ribozymes, phosphatase inhibitors or SH2/SH3-directed agents). Selected tyrosine kinase inhibitors, known or believed to be in development in cancer treatment trials, are summarized as are some of the key issues that must be addressed if these compounds are to be developed into clinically useful cancer chemotherapeutic agents.

Halogenated L-fucose and D-galactose analogues: synthesis and metabolic effects.

Several new analogues of L-fucose modified in the 2 position and the 5-methyl group have been synthesized as potential plasma-membrane glycoconjugate inhibitors or modifiers, and their biological effects have been studied. 2-Chloro-, 2-bromo-, and 2-iodo-2-deoxy-L-fucose (9a, 9b, and 13, respectively) have been prepared by addition of the appropriate halogen to 3,4-di-O-acetyl-L-fucal, followed by hydrolysis of the anomeric halogen and the acetyl groups. A series of four halogenated 5-methyl analogues of L-fucose (4, X = F, Cl, Br, and I) have been obtained starting from 1,2:3,4-di-O-isopropylidene-L-galactose. The synthesis of this latter compound has been improved. A corresponding series of 6-deoxy-6-halo-D-galactose analogues, which are enantiomers of the 5-(halomethyl)-L-fucose analogues, has also been synthesized. Analogues 4b, 4c, and 9b at 1 x 10(-3) M specifically inhibited the incorporation of L-[3H]fucose into macromolecular components of SW613 human mammary tumor cells. Analogue 13 inhibited the growth of L1210 murine leukemic cells with an IC50 of 6 x 10(5) M in culture. 6-Deoxy-6-fluoro-D-galactose and its enantiomer 4a were found to be effective inhibitors of D-[3H]galactose and L-[3H]fucose incorporation, respectively, into macromolecular components of human mammary tumor cells. The effectiveness of inhibition was reduced with an increase in size of the halogen atom. Analogue 4a and its enantiomer have been tritiated at C-1 and both were found to be activated to a nucleotide sugar, which was followed by incorporation into the macromolecular fraction of SW613 human mammary tumor cells in vitro.

Potentiation of butyrate-induced differentiation in human colon tumor cells by deoxycholate.

Human colon adenocarcinoma cells, treated with deoxycholate for 24 h prior to exposure to 1 mM butyrate, exhibited dose-dependent increases in the activities of three markers of colonic differentiation (alkaline phosphatase, lactase and CEA). Treatment with deoxycholate alone, for 24 h or longer, did not increase the secretion of CEA or the activities of either of the brush border-associated enzyme activities. Increases in differentiation markers were found to be bile acid-specific. Pretreatment with either dehydrocholic acid or cholic acid, even at cytotoxic concentrations, led to no significant butyrate-induced increases in brush-border associated hydrolase activities. The addition of a bacterial superoxide dismutase decreased the short-term cytotoxicity of deoxycholate and increased the maturation-potentiating effects of the bile acid in HCT-116 DO cells. The results of these studies demonstrate that bile acids, which are commonly thought to have tumor promoting activities in vivo, may also have physiological effects which serve to limit carcinogenic processes in the human colon by potentiating tumor cell differentiation.

Effects of a membrane sugar analogue, 6-deoxy-6-fluoro-D-galactose, on the L1210 leukemic cell ectosialyltransferase system.

In L1210 leukemia cells, 6-deoxy-6-fluoro-D-galactose specifically inhibited the incorporation of [3H]-D-galactose, while that of other precursors of glycoconjugate biosynthesis, including mannose and glucosamine, was unaffected. The activation of [6-3H]-6-deoxy-6-fluoro-D-galactose to a nucleotide sugar was similar to that found for [3H]-D-galactose. The incorporation of either sugar after 1 hr was visualized by electron microscopic autoradiography to be in the Golgi region. Treatment of L1210 cells with 6-deoxy-6-fluoro-D-galactose in vitro or in vivo resulted in a specific, dose- and time-dependent decrease in the activity of cell surface sialyltransferase (ectosialyltransferase) but not of 5'-nucleotidase, a plasma membrane marker enzyme. The decrease in ectosialyltransferase activity appeared to be selective and is suggested to be due to structural modification of the cell surface galactoprotein acceptors for this enzyme. The data indicate that 6-deoxy-6-fluoro-D-galactose is an effective modifier of cellular glycoconjugate in that its incorporation into certain cell surface components results in a modification of plasma membrane structure and function.

Mechanisms involved in the induction of malignant cell differentiation.

Cancer appears to be a disease of altered maturation, with changes in genetic expression leading to a situation in which the physiological regulation of cellular proliferation and maturation are altered. Environmental factors as well as defined chemical agents have been demonstrated to have the capacity to convert neoplastic cells to end-stage forms with a finite life span through a process characteristic of cellular maturation. The correction of genetic defects by these inducers of differentiation does not appear to be required; the critical feature is that the differentiated cells assume a state in which they no longer possess the capability for continued cellular replication. The extrapolation of these advances, accomplished in experimental systems, to clinical practice should yield significant decreases in the neoplastic cell burden without the degree of morbidity produced by aggressive therapy with cytodestructive agents, especially when employed in multidrug combinations. The ultimate introduction of differentiation as a therapeutic approach to cancer treatment if attained, however, will require a variety of principles to be established, so that optimum efficacy may be obtained from each agent, the fabrication of new agents with major changes in the ratio of the concentrations required to produce cytotoxicity relative to those necessary to initiate maturation is attained, and the elucidation of non-antagonistic combinations of differentiation inducing agents with or without cytotoxic drugs is achieved to combat the problem of tumor cell heterogeneity.

Rapid increase in the activity of DNA topoisomerase I, but not topoisomerase II, in HL-60 promyelocytic leukemia cells treated with a phorbol diester.

There is significant evidence to suggest that protein kinase C and DNA topoisomerases are functionally linked in signal transduction pathways. Much of this is based on the observation that phosphorylation of topoisomerase II by protein kinase C may lead to its activation in vitro and that inhibitors of topoisomerase II block phorbol diester-induced differentiation in HL-60 cells. In the present study, the activities of the DNA topoisomerases I and II have been quantitated to examine their regulation in phorbol diester-treated HL-60 cells undergoing differentiation. The activity of topoisomerase I increased rapidly after treatment with phorbol myristate acetate (PMA); it increased maximally (150% of control activity) at 3 hr post-treatment and remained elevated for at least 24 hr. Conversely, from the onset of exposure to PMA through 12 hr, there was no measurable alteration in topoisomerase II activity in PMA-treated cells. Moreover, there was a measurable decrease in topoisomerase II activity at the later time points, a result that occurred concomitantly with the loss of proliferative potential in differentiating HL-60 cells. Similar results were obtained when the activities of both enzymes were measured in nuclear extracts. The apparent increase in topoisomerase I activity was not due to an increase in the mass of the enzyme after PMA treatment, as measured by both western blotting and by the formation of camptothecin-dependent, topoisomerase I-DNA complexes. Taken together, these data suggest that the activities of the topoisomerases I and II may have been regulated independently in PMA-treated HL-60 cells, that the activity of topoisomerase II was not increased under conditions in which protein kinase C was activated in vivo, and that an increase in the activity of topoisomerase I may have had a role in the mechanism through which HL-60 cells underwent monocytic maturation in response to phorbol diesters.

Increased cell surface EGF receptor expression during the butyrate-induced differentiation of human HCT-116 colon tumor cell clones.

Several clonal sublines of HCT-116 human colon adenocarcinoma cells were isolated and characterized on the basis of their growth characteristics, intrinsic enterocyte-like differentiation (as assessed by alkaline phosphatase and lactase activities), and responses to butyrate, an inducer of colon tumor cell maturation. The HCT-116 sublines were found to be heterogeneous and several phenotypically distinct clones were identified. Further characterization of these clones indicated that the effects of butyrate on cell growth, alkaline phosphatase activity, and lactase activity were distinct and separable. The growth of all of the clones were inhibited by butyrate (IC50 values varied from 0.44 to 1.5 mM), but the effects of this agent on alkaline phosphatase and lactase activities varied widely. In several sublines butyrate had no effect on either enzyme while in others one or both activities were induced. Additionally, the binding of 125I-epidermal growth factor (EGF) to cell surface receptors was found to be proportional to the expression of lactase activity in the cell. The D3 clone and other sublines with intrinsic lactase activities greater than 100 nmol/mg/min expressed a class of high-affinity EGF receptors (e.g., D3 cells had 3.48 X 10(4) EGF receptors/cell with a kd of 0.61 nM). Other clones with less lactase activity had undetectable levels of 125I-EGF binding. In clones which exhibited greater than twofold increases in lactase activity in response to butyrate, the expression of a large number of low-affinity EGF receptors was also induced. In one such clone, the P1 subline, lactase activity was increased from 70 nmol/mg/min to 230 nmol/mg/min after 96 h in 2 mM butyrate, and the expression of EGF receptors was increased from undetectable levels to 1.18 X 10(5) EGF receptors/cell (kd of 3.2 nM). Northern blot analysis indicated that the increased 125I-EGF binding after butyrate treatment may have been due, in part, to a greater than twofold accumulation of EGF receptor mRNA. In addition, the expression of the messages for transforming growth factor alpha (TGF-alpha) and transforming growth factor beta (TGF-beta) was examined in butyrate-treated cells. While TGF-alpha mRNA levels were found to correlate with EGF receptor message levels in the HCT-116 clones, TGF-beta mRNA expression was not found to correlate with the butyrate-induced growth inhibition or with increases in EGF receptor expression, alkaline phosphatase activity, or lactase activity in these cells.

Dissociation of protein kinase C activation from phorbol ester-induced maturation of HL-60 leukemia cells.

The role of C-kinase in the induction of maturation of HL-60 promyelocytic leukemia cells was examined using two activators of this kinase, 12-O-tetradecanoyl phorbol 13-acetate (TPA) and 1-oleoyl-2-acetylglycerol (OAG). At 10(-8) M, a concentration that induced maturation, TPA effectively stimulated C-kinase activity in cell-free preparations by increasing the affinity of the enzyme for Ca2+. Similar activation was observed with 20 micrograms/ml of OAG. At these concentrations, addition of either compound to intact cells stimulated the phosphorylation of cellular proteins. Treatment with TPA resulted in an increased phosphorylation of 14 proteins, 9 of which also changed in response to OAG. In addition to the effects on protein phosphorylation, TPA and OAG both affected choline lipid metabolism. TPA at 10(-8) M stimulated the incorporation of [methyl-3H]choline into phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine. OAG at 20 micrograms/ml had quantitatively similar effects on the labeling of the former two lipids, but did not affect incorporation of choline into lysophosphatidylcholine. Despite the similar biochemical effects of TPA and OAG, the diglyceride was unable to induce HL-60 cell maturation as measured by inhibition of cell growth, development of nonspecific esterase activity, phagocytosis, adherence of cells to plastic, and loss of transferrin receptor activity. The lack of effect is not due to metabolism of OAG; maturation could not be induced by treating cells with fresh OAG every 2 h for a period of 12 h. These results suggest a dissociation of the activation of C-kinase and the induction of HL-60 cell maturation by TPA.

Reovirus infection in rat lungs as a model to study the pathogenesis of viral pneumonia.

We undertook the present study to elucidate the pathogenesis of the pathologic response to reovirus infection in the lungs and further understand the interactions of reoviruses with pulmonary cells. We found that reoviruses were capable of causing acute pneumonia in 25- to 28-day-old Sprague-Dawley rats following intratracheal inoculation with the reoviruses type 1 Lang (T1L) and type 3 Dearing (T3D). The onset of the pneumonia was rapid, marked by type I alveolar epithelial cell degeneration, type II alveolar epithelial cell hyperplasia, and the infiltration of leukocytes into the alveolar spaces. More neutrophils were recruited into the lungs during T3D infection than during T1L infection, and the serotype difference in the neutrophil response was mapped to the S1 gene of reovirus. Viral replication in the lungs was required for the development of pneumonia due to T1L and T3D infections, and replication occurred in type I alveolar epithelial cells. T1L grew to higher titers in the lungs than did either T3D or type 3 clone 9, and the S1 gene was found to play a role in determining the level of viral replication. We propose that experimental reovirus infection in the lungs can serve as a model for the pathogenesis of viral pneumonia in which pulmonary inflammation results following direct infection of lung epithelial cells.

The biochemical and ultrastructural effects of tunicamycin and D-glucosamine in L1210 leukemic cells.

Tunicamycin was found to specifically inhibit the incorporation of a number of sugars into L1210 leukemia cell glycoproteins. This inhibition of glycoprotein biosynthesis led to a cessation of cell growth which was reversible in a dose-dependent and time-dependent manner. After removal of the antibiotic from L1210 cell cultures resumption of sugar incorporation preceded that of thymidine incorporation and the recovery of cell growth. The treatment of cells with tunicamycin resulted in a significant increase in the intracellular pool of UDP-N-acetylglucosamine which occurred concurrently with alterations in cell ultrastructure including distentions of the endoplasmic reticulum and nuclear membranes. Similar ultrastructural changes and increases in the intracellular pools of UDP-sugars were observed in L1210 cells exposed to 5 mM D-glucosamine, which suggested that the antiproliferative effects of tunicamycin may be related to the accumulation in the endoplasmic reticulum of one or more nucleotide sugar precursors of asparagine-linked glycoprotein biosynthesis. However, the biological effects of tunicamycin could be distinguished from those caused by D-glucosamine. Exposure of L1210 cells to tunicamycin resulted in specific alterations in the biochemical composition of the plasma membrane and in the inhibition of cellular agglutination by wheat germ agglutinin which were not apparent following exposure to equitoxic concentrations of the aminosugar. These studies, together with those which demonstrated that recovery of the cellular capacity to synthesize glycoproteins was obligatory for the recovery of cellular proliferation in tunicamycin-treated cells, suggested that inhibition of the synthesis of glycoproteins was the major factor limiting L1210 leukemic cell proliferation.

Phorbol diester-induced alterations in the expression of protein kinase C isozymes and their mRNAs. Analysis in wild-type and phorbol diester-resistant HL-60 cell clones.

In an HL-60 cell subline (PR-17) which was greater than 100-fold resistant to the differentiating and cytostatic activities of phorbol 12-myristate 13-acetate (PMA), the protein kinase C phenotype was found to be nearly identical to that of wild-type HL-60 cells. A measurable decrease (30%) in the specific activities of crude preparations of PR-17 cell protein kinase C was observed when the enzyme was measured with histone as the phosphate acceptor substrate, but other aspects of the protein kinase C phenotype (intracellular concentrations and binding affinities of phorbol diester receptors, translocation of activated enzyme from cytosolic to particulate subcellular fractions, relative expression of the alpha and beta isozyme proteins) were equivalent in both PMA-resistant PR-17 cells and in wild-type HL-60 cells. Direct analysis of the behavior of the alpha and beta isozymes after the exposure of each cell type to 100 nM PMA for 12 h revealed that the activities and intracellular concentrations of both isozymes were downregulated to an equivalent extent in both wild-type and PMA-resistant cells. These results suggest that the cellular basis for the resistance to the effects of PMA was present "down-stream" from the activation and down-regulation of protein kinase C and was perhaps a nuclear component. Among the genes which were likely to be differentially regulated when each of the two cell lines were treated with PMA were those for the protein kinase C isozymes themselves. In wild-type HL-60 cells, the intracellular concentrations of type HL-60 cells, the intracellular concentrations of mRNA for each of the beta isozymes were increased (up to 5-fold) 48 h after the initiation of PMA treatment; further studies indicate that an activator of protein kinase C could influence the expression of HL-60 cell protein kinase C genes in an isozyme-specific manner. Comparable PMA-induced alterations in mRNA levels were not observed in PMA-resistant cells, even under conditions of significant activation and subsequent down-regulation of protein kinase C protein. Taken together, these data suggest that activation and down-regulation of the isozymes of protein kinase C may not represent absolute determinants of the PMA-induced differentiation of HL-60 cells, but that specific alterations in the levels of the mRNA for the beta isozymes of protein kinase C, or of other genes which may be regulated by the activated kinase isozymes, are important to the induction of leukemia cell differentiation by PMA.

Antibody-forming cell response to virus challenge in mice immunized with DNA encoding the influenza virus hemagglutinin.

Immunization of mice with DNA encoding the influenza virus hemagglutinin (HA) affords complete protection against lethal influenza virus infection and the means to investigate the mechanisms of B-cell responsiveness to virus challenge. Using a single-cell enzyme-linked immunospot assay, we sought to determine the localization of HA-specific antibody-forming cells (AFCs) during the development of humoral immunity in mice given HA DNA vaccine by gene gun. At 33 days postvaccination, populations of AFCs were maintained in the spleen and bone marrow. In response to lethal challenge with influenza virus, the AFCs became localized at the site of antigenic challenge, i.e., within the draining lymph nodes of the lung compartment. Immunoglobulin G (IgG)- and IgA-producing AFCs were detected in lymph nodes of the upper and lower respiratory tracts, underscoring their importance in clearing virus from the lungs. Response to challenge required competent CD4+ T cells, without which no AFCs were generated, even those producing IgM. By contrast, in mice vaccinated with an HA-containing subunit vaccine, fewer AFCs were generated in response to challenge, and these animals were less capable of resisting infection. Our findings demonstrate the comparable localization of AFCs in response to challenge in mice vaccinated with either HA DNA or live virus. Moreover, the former strategy generates both IgG- and IgA-producing plasma cells.

Differential expression of inducible nitric oxide synthase messenger RNA along the longitudinal and crypt-villus axes of the intestine in endotoxemic rats.

OBJECTIVES: To characterize the mechanisms leading to excessive production of nitric oxide within the gut as a consequence of endotoxemia. We sought to: a) determine the time course of inducible nitric oxide synthase (iNOS) messenger RNA (mRNA) expression in the intestine after challenging rats with lipopolysaccharide (LPS); and b) investigate whether there is differential expression of iNOS in enterocytes along the longitudinal or crypt-villus axes of the intestine in rats after LPS administration. DESIGN: Prospective, randomized, unblinded study. SETTING: Research laboratories at a large university-affiliated medical center. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: At T = 0 hr, rats were injected with O111:B4 Escherichia coli LPS (5 mg/kg) or a similar volume of the saline vehicle. At various time points thereafter, samples of duodenum, jejunum, ileum, colon, and liver were harvested for subsequent extraction of RNA. In some cases, populations of enterocytes enriched in either crypt or villus cells were harvested from the ileum. In some studies, rats were injected with cycloheximide (25 mg i.p.) 15 mins before being challenged with LPS or dexamethasone (2 mg i.p.) 30 mins before being injected with LPS. MEASUREMENTS AND MAIN RESULTS: iNOS mRNA was undetectable in ileal tissue from rats under basal conditions, but was evident by T = 1 hr and was maximal at T = 2 hrs after injection of LPS. Thereafter, ileal iNOS mRNA concentrations decreased and were undetectable again at T = 24 hrs. At T = 2 hrs after LPS injection, there was marked expression of iNOS mRNA in the ileum, whereas much lower concentrations of iNOS mRNA were detected in the jejunum and colon, and no iNOS mRNA was detected in the duodenum. At T = 3 hrs after LPS injection, expression of iNOS mRNA was up-regulated in both villus and crypt cells, although LPS-induced iNOS mRNA was more prominent in the former than the latter cell type. Pretreatment of rats with dexamethasone virtually abrogated the expression of iNOS mRNA in ileal samples obtained 3 hrs after the injection of LPS. Prior treatment of rats with the protein synthesis inhibitor, cycloheximide, also blunted LPS-induced iNOS mRNA expression. CONCLUSIONS: LPS-induced iNOS expression is differentially regulated along both the longitudinal and crypt villus axes of the intestinal mucosa, being most prominent in the villus cells of the ileum. LPS-induced iNOS expression is blunted by pretreating rats with dexamethasone or cycloheximide. The latter finding suggests that LPS-induced expression of iNOS mRNA in the gut requires new protein synthesis. Differential regulation of nitric oxide production along the longitudinal and crypt-villus axes of the gut may be a determinant of the pattern of sepsis-induced intestinal damage.