Effects of interferon gamma on growth, apoptosis, and MHC class II expression of immature rat intestinal crypt (IEC-6) cells.

Intestinal epithelial cells and the mucosal immune cells in close proximity are thought to interact very closely. One well-established mechanism of this intercellular cross-talk is via the production of cytokines such as interferon gamma (IFNgamma). The aim of this study was to analyze the effects of IFNgamma on intestinal crypt epithelial cells. IEC-6 cells were cultured in the presence or absence of IFNgamma to measure its effects on proliferation, cell cycle, apoptosis, and major histocompatibility complex (MHC) class II antigen expression. Even at very low doses (0.01 U/ml), IFNgamma significantly inhibited IEC-6 cell proliferation, as demonstrated by reduced 3H-thymidine uptake, stable cell count, and complete arrest in the quiescent G0/G1 phase of the cell cycle. Incubation with supraphysiological doses of IFNgamma (100-1,000 U/ml) did not induce apoptosis, as assessed by morphology and the TUNEL assay. IFNgamma significantly induced de novo IEC-6 class II antigen expression. Tumor necrosis factor alpha (TNF alpha), which alone had no effect, synergistically enhanced this effect of IFNgamma. MHC class II antigen expression was observed to be independent of cell cycle phase. Our results indicate that IFNgamma alters immature crypt epithelial cell turnover and upregulates MHC class II expression. These alterations may be important in the pathogenesis of immune-mediated bowel disorders.

Distinct patterns of cow's milk allergy in infancy defined by prolonged, two-stage double-blind, placebo-controlled food challenges.

BACKGROUND: The clinical manifestations of cow's milk allergy (CMA) are highly variable, and challenges usually identify only immediate, IgE mediated reactions. OBJECTIVE: To clearly identify CMA of immediate and delayed types using a two-stage, double-blind, placebo-controlled food challenge (DBPCFC), and to prospectively compare the clinical history and analyses of specific IgE antibodies to milk in predicting outcome of DBPCFC. METHODS: A total of 69 patients (33 girls, 36 boys) were recruited for study based on a history highly suggestive of CMA and resolution of symptoms on a bovine protein-free diet. After skin-prick tests (SPTs) and search for allergen-specific serum IgE antibodies by enzyme allergosorbent test (EAST), a two-stage DBPCFC was performed over several days. RESULTS: Of 16 patients (mean age 36.9 months) classified as probable immediate reactors based on the history, 10 (62.5%) had a positive DBPCFC with similar patterns to historical adverse reactions (< or = 2 h after milk exposure). The other 53 (77%) patients (17.3 months) had a history of probable delayed type CMA presenting with predominantly gastrointestimal symptoms from 2 h and up to 6 days after milk exposure. Of these, 15 (28.8%) had a positive DBPCFC, again with a symptom pattern similar to the history. Sensitivity/specificity of SPT was similar to that of EAST for both immediate (70/83% and 62/83% respectively, NS) or delayed (0/97% and 0/97%) CMA confirmed by DBPCFC. CONCLUSIONS: Using our two-stage, prolonged DBPCFC, we clearly identified two groups of children with CMA, reflecting different pathogenesis of either immediate-type IgE-dependent, or delayed-type IgE-independent allergy. Although useful in immediate reactors, IgE antibody determination cannot predict the outcome of DBPCFC in delayed reactors. A thorough clinical history was the most helpful tool to predict the type of response in challenge positive patients.

Tumor necrosis factor-alpha inhibits lipid and lipoprotein transport by Caco-2 cells.

Cytokines, important mediators of inflammation, have been shown to cause disturbances in circulating and hepatic lipid metabolism. Although the intestine plays a major role in dietary fat transport and largely contributes to plasma lipoproteins, the effects of cytokines on intestinal lipid handling remain unknown. In the present study, the modulation of lipid, apoprotein, and lipoprotein synthesis and secretion by tumor necrosis factor-alpha (TNF-alpha) was investigated in Caco-2 cells. Highly differentiated and polarized cells (20 days in culture) were incubated for 20 h with recombinant human TNF-alpha (100-500 ng/ml). No cytotoxic effect of TNF-alpha cells was observed, as indicated by the determinations of Caco-2 cell viability and monolayer transepithelial resistance. Moreover, no differences in cell maturation (sucrase activity) or cell proliferation ([3H]thymidine incorporation and cell cycle analysis) were detected between treated and control cultures. Significant inhibition of lipid secretion by TNF-alpha was observed, with the greatest reduction at 500 ng/ml. TNF-alpha significantly decreased Caco-2 cell secretion of phospholipids (22%), triglycerides (30%), and cholesteryl ester (37%). It also significantly diminished the export of newly synthesized low-density lipoproteins (LDL; 20%) and high-density lipoproteins (HDL; 13%), with a lesser effect on very low-density lipoproteins (VLDL; 3%). The lipid composition of these lipoproteins was minimally affected. De novo synthesis of apo A-I, apo B-100, and apo B-48 was also markedly reduced by TNF-alpha. Sphingomyelinase activity was not increased and cell content of sphingomyelin was not altered, suggesting that inhibitory effects on lipid and apoprotein of TNF-alpha were not mediated by the ceramide pathway. Our results indicate that TNF-alpha may play a role in modulating intestinal lipid metabolism, thus affecting circulating lipoproteins.

Lipid esterification and synthesis by the IEC-6 intestinal epithelial crypt cell line: effect of transforming growth factor beta.

The capacity of immature intestinal epithelial crypt cells to synthesize lipids and the factors that promote their differentiation remain largely unknown. We examined the profile of lipids synthesized by a normal rat intestinal epithelial crypt cell line (IEC-6) and determined the effects of transforming growth factor beta (TGF beta), a putative crypt cell differentiating factor, on their lipid handling. Incubation of IEC-6 cells with [14C]oleic acid (20 h) resulted in lipid esterification and synthesis, mainly as triglycerides (TGs, 57 +/- 0.6%) and phospholipids (PLs, 30 +/- 0.6%), with a PL/TG ratio of 0.53. When cells were pulsed (2.5 h) with [14C]oleic acid and then maintained 20 h in medium alone, a significant elevation of the PL/TG ratio (10.2 +/- 1.3, p < 0.01) was observed, primarily accounted for by a significant decrease of the TG fraction (p < 0.01). IEC-6 cells secreted only trace amounts of lipids under the latter experimental condition. Incubation with TGF beta (20 h) significantly inhibited IEC-6 cell proliferation but did not promote the expression of cell sucrase activity. TGF beta induced a significant increase in the cellular composition of PL (p < 0.05) and a decrease in the TG fraction (p < 0.02), after a 2.5-h pulse of [14C]oleic acid. Lipid production was unaffected by TGF beta during the 20-h incubation with [14C]oleic acid. Lipid secretion into the medium remained negligible in the presence of TGF beta, after 2.5 h of incubation with substrate as above. Our findings suggest that immature crypt IEC-6 cells are capable of lipid esterification and synthesis but secrete minute amounts of lipoproteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-2 production in pediatric inflammatory bowel disease: evidence for dissimilar mononuclear cell function in Crohn's disease and ulcerative colitis.

Crohn's disease (CD) and ulcerative colitis (UC) represent clinically distinct chronic inflammatory bowel diseases (IBD) of unknown etiology. Although the mucosal immune system is implicated in their pathogenesis, immunological differences between the two disorders are not well defined. The aim of this study was to compare in vitro mucosal T-lymphocyte function in CD and UC. Peripheral blood mononuclear cell interleukin-2 (IL-2) production was similar in pediatric IBD and control patients under unstimulated conditions, but was significantly increased in response to phytohemagglutinin (PHA) stimulation for the UC group. Lamina propria mononuclear cells (LPMNC) isolated from colonic resections in IBD patients had significantly lower spontaneous proliferation and IL-2 production in vitro than did LPMNC of control patients. In contrast, significantly greater IL-2 production was detected when the LPMNC of CD patients were cultured with PHA, in comparison with those of UC or control patients. When indomethacin, a prostaglandin synthetase inhibitor, was added to the cultures, significantly increased IL-2 secretion was observed for CD LPMNC, but not for UC cultures, under both stimulated and unstimulated conditions. These findings demonstrate abnormal LPMNC IL-2 production in IBD. Furthermore, our data suggest that inhibition of the prostaglandin synthetase pathway upregulates IL-2 production by LPMNC in CD. These results support the hypothesis that immunoregulatory mechanisms controlling IL-2 production differ between CD and UC.

Circulating tumor necrosis factor-alpha levels and lipid abnormalities in patients with cystic fibrosis.

Hyperlipidemia is prominent among the disturbances in intermediary metabolism that occur subsequent to infections by microorganisms. The response to such infections is known to involve several cell types and is mediated by cytokines. We hypothesized that metabolic lipid disturbances seen during infection in cystic fibrosis (CF) patients may partly be the result of excessive tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine known to cause a large spectrum of pathophysiologic alterations, including impaired lipid metabolism. Therefore, we determined the circulating concentration of TNF-alpha and analyzed its relationship to lipid and lipoprotein levels, as well as lipoprotein lipase activity, in 31 CF patients. Plasma TNF-alpha values were significantly (p < 0.01) elevated in patients with CF compared with controls. The CF subjects were found to have decreased plasma cholesterol (25%), LDL cholesterol (35%), and HDL cholesterol (19%) concentrations, whereas plasma triglycerides were significantly increased (p < 0.001). The apo A-I level was reduced (p < 0.005), whereas apo B levels were normal. Low levels of the major essential fatty acids were found in the plasma of the CF patients, and the triene/tetraene ratio confirmed their essential fatty acid deficiency. Postheparin lipolytic activity was lower in CF patients than in controls, and the decreased activity was accounted for primarily by a decline in hepatic lipase. A significant positive correlation (p < 0.001, r = 0.70) was found between TNF-alpha and plasma triglyceride levels. However, no association was noted between TNF-alpha and essential fatty acid, cholesterol, or lipoprotein cholesterol levels, or with lipoprotein lipase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Beneficial effects of fish-oil supplements on lipids, lipoproteins, and lipoprotein lipase in patients with glycogen storage disease type I.

Glycogen storage disease type I (GSD-I) is frequently complicated by severe hyperlipoproteinemia and the increased potential risk of premature atherosclerosis. The effects of fish-oil supplementation [MaxEPA, 10 g.(1.73 m2)-1 for 3 mo] were investigated prospectively in seven hyperlipoproteinemic patients with GSD-I. Hypertriglyceridemia and hypercholesterolemia improved after 3 mo of fish-oil treatment, decreasing 49% (P < 0.005) and 23%, respectively. This was accompanied by a reduction in both low-density-lipoprotein (LDL) cholesterol (25%, P < 0.03) and apolipoprotein B (40%) and by increased high-density-lipoprotein increased (HDL) cholesterol (30%, P < 0.002) and apolipoprotein A-I (31%, P < 0.05). Low pretreatment ratios of HDL to total cholesterol and HDL to LDL, indicators of elevated atherosclerosis risk, increased significantly (P < 0.05). Plasma lipoprotein profile as well as lipoprotein composition [triglyceride (TG) enrichment and cholesteryl depletion] improved. Reduced TG concentrations were due to enhanced fat catabolism, as evidenced by the significantly increased hepatic and extrahepatic lipoprotein lipase activity (P < 0.05). Withdrawal of fish oil for 3 mo was associated with a return to pretreatment abnormalities in plasma lipids and lipoproteins. Fish-oil supplementation thus improves the hyperlipoproteinemia in GSD-I and may significantly reduce the risk of premature atherosclerotic cardiovascular disease.

Interleukin-2 activity of colonic lamina propria mononuclear cells in a rat model of experimental colitis.

BACKGROUND: Altered interleukin 2 (IL-2) production has been implicated in the pathogenesis of inflammatory bowel diseases. METHODS: The temporal relationship between IL-2, prostaglandin E2 (PGE2) production, and mucosal injury was evaluated by isolated colonic lamina propria mononuclear cells (LPMC), using the trinitrobenzene sulfonic acid model of rat colitis. RESULTS: Spontaneous LPMC IL-2 activity was significantly increased in chronic (5 weeks) but not acute (5 days) or resolved colitis groups. IL-2 activity after concanavalin A activation was highest in the groups with resolved and chronic colitis. PGE2 production was significantly increased in LPMC cultures in acute or chronic colitis as well as the ethanol control groups but not the resolved colitis group. The addition of indomethacin to LPMC cultures decreased PGE2 levels in all groups, whereas IL-2 activity increased only for the chronic and resolved colitis groups. No correlation was found between PGE2 and IL-2 production by LPMC. CONCLUSIONS: In this experimental model, LPMC IL-2 production varied according to the severity and duration of the inflammation. Increased PGE2 production does not appear to be responsible for the IL-2 alterations in colitis.

Olive (Olea europea) pollen allergens--I. Immunochemical characterization by immunoblotting, CRIE and immunodetection by a monoclonal antibody.

The pattern of reactivity of the Olea europea crude extract antigens was analysed after electroblotting to nitrocellulose from SDS-PAGE. The antigens contained in the 17, 19 and 42 K bands were most reactive with specific IgE from individual sera. Following immunization with a crude extract, one monoclonal antibody (OL-1) was raised against components which exhibited IgE binding capacity in electroblotting and crossed radioimmunoelectrophoresis (CRIE). Monoclonal antibody OL-1 reacted with the 17 and 19 K antigens and with three arcs of crossed immunoelectrophoresis (CIE), one of which is considered to contain a major allergen by CRIE.

T cell dependence of human IgE response: quantitative and functional studies.

In 108 allergic and nonallergic patients T3, T4 and T8 cells were quantified. No significant differences were found when comparing healthy and allergic individuals. Functionally, the role of T8 suppressor and cytotoxic population was studied by removing this subset, comparing the IgE produced in vitro by peripheral blood lymphocytes of unfractionated and T8-depleted cultures. Results indicate a statistically significant increase of the in vitro IgE production when cultures from healthy people and 'normoproducer' allergics (in vitro spontaneous IgE production no more than 900 pg/ml/10(7) cells) were depleted of T8 cells. The same experiments in 'hyperproducer' allergics (in vitro spontaneous IgE production more than 900 pg/ml/10(7) cells) show no significant difference in the IgE production when T8 cells were depleted.

Stability of Lolium perenne extract.

The stability of Lolium perenne extract was studied during a year in different conditions of storage. The changes of allergen activity were measured periodically by both in vivo and in vitro techniques, and structural modifications were examined by polyacrylamide gel electrophoresis and isoelectric focusing. Lyophilised extracts did not show any change and frozen samples retained full activity, but there were slight alterations in the pattern of proteins during the storage period. The activity of refrigerated aqueous extracts (4-6 degrees C) decreased gradually with time, while glycerinated samples at the same temperatures did not lose any of their allergen potency. Room temperature and 40 degrees C were unsatisfactory for aqueous extracts, but less so for preparations that contained 50% glycerol. The presence of the preservative phenol had a significant negative effect on stability at all of the temperatures studied.

Transmethylation reactions in human basophils induced by anti-IgE or specific antigen.

The activation of methyl transferases in the membrane of human basophilic leukocytes was measured by incorporation of 3H-methyl groups into phospholipids. Basophils of healthy individuals were activated by anti-IgE, those of pollen allergic patients by the specific antigen, Lolium perenne extract. Basophil-rich fraction was obtained from peripheral blood by centrifugation in a gradient of Ficoll/metrizamide (g = 1.085). After incubation with anti-IgE an increase in the 3H-methyl incorporation was detected during the first 30 s of the reaction. The maximal incorporation in basophils of the pollen allergic patients, activated by L. perenne extract, was enhanced at 15 s. Appropriate controls showed that the activation of methyl transferases occurred in basophils only.

Fractionation and biological characterization of Olea europea pollen extract.

Preliminary fractionation of Olea european pollen extract has been performed. At least 10 antigenic fractions have been found by crossed electrophoresis. After Sephadex gel filtration, two fractions with a molecular weight of 160,000 and 65,000 have been obtained. The fractions were evaluated for allergenic activity by two in vitro techniques: polystyrene tube radioimmunoassay (PTRIA) and basophil degranulation test (BDT), and by skin tests. All tests indicated that the most reactive fractions were those in the 65,000 molecular weight peak. BDT has been shown to be a very reliable method as compared with histamine release and other parameters. Although common antigenic fractions have been found for Lollium perenne and O. europea, no cross-allergenicity has been shown by PTRIA inhibition.