Eduard F. Stange

Steroid-unresponsive acute attacks of inflammatory bowel disease: immunomodulation by tacrolimus (FK506)

Objective:Steroid treatment failure in acute Crohns disease and ulcerative colitis frequently necessitates surgical intervention. Several alternative therapeutic strategies have been raised. The most promising so far has been intravenous cyclosporine, but the results in the long term have been discouraging. We assessed the efficacy and safety of the new macrolide immunomodulator tacrolimus as an alternative to cyclosporine A.Methods:Eleven patients with steroid-refractory disease (six ulcerative colitis, two indeterminate colitis, two Crohns disease, one pouchitis) and severe activity according to the Truelove and Witts criteria or Crohns disease activity index > 150, respectively, were eligible for the study. All patients were treated with intravenous tacrolimus for 7–10 days followed by oral treatment over a median period of 7 months (range 0.25–16). Azathioprine and mesalamine were given concomitantly. Steroids were tapered according to clinical activity.Results:Seven of 11 patients achieved remission rapidly, whereas a modest improvement was noted in two. Only two patients required an early and one a delayed colectomy. Moreover, a rectovaginal fistula closure in a case of Crohns disease and an improvement of pouchitis was observed. A tapering to low dose steroids was possible during oral tacrolimus therapy in all nine responders and remission was maintained in five of them (mean follow-up 9.2 months). The drug was well tolerated and side effects were managed conservatively.Conclusion:Tacrolimus induced rapid remission in steroid resistant inflammatory bowel disease in the majority of cases. It appears to be an effective treatment modality that may be superior to cyclosporine with respect to maintenance of remission.

Mesenteric blood flow is related to disease activity and risk of relapse in Crohn's disease: a prospective follow-up study

Objective:The diagnostic significance of increased splanchnic blood flow in Crohns disease is unclear. This prospective study was therefore undertaken to define the role of Doppler sonography in the assessment of disease activity and in the prediction of early relapse.Methods:Splanchnic flowmetry was performed in 59 patients with Crohns disease and 20 healthy volunteers during fasting and 30 min after ingestion of a standardized meal. Twenty-one patients measured during the active state and in clinical remission were followed-up for 6 months. Hemodynamic parameters of the superior and inferior mesenteric arteries and the portal vein were related to clinical (Crohns disease activity index [CDAI]), laboratory (C-reactive protein), and endoscopic (Crohns Disease Endoscopic Index of Severity) parameters of disease activity.Results:The postprandial mean velocity of the superior mesenteric artery correlated closest with clinical activity (CDAI, p < 0.005) and C-reactive protein (p < 0.01), but was unrelated to endoscopic activity. All patients in remission after 6 months (9/9) showed an increase in postprandial pulsatility index of the superior mesenteric artery, compared with an initial measurement during active disease (+28%). In contrast, the majority of patients with later relapse or surgery (11/12) had decreased pulsatility index during initial remission (−20%). The positive predictive value of this index for maintenance of remission was 0.82.Conclusion:Postprandial flow measurements in the superior mesenteric artery are closely related to clinical but not endoscopic disease activity in patients with Crohns disease. The repeated measurement of the postprandial pulsatility index allows estimation of the risk of recurrence.

The postprandial portal flow is related to the severity of portal hypertension and liver cirrhosis

BACKGROUND/AIMS Diminished postprandial portal hyperemia has been demonstrated by echo-Doppler flowmetry in patients with liver cirrhosis, but its diagnostic role is unclear. This prospective study was therefore undertaken in patients with varying severity of portal hypertension and degree of liver cirrhosis. METHODS Portal flowmetry was performed in 66 patients with cirrhosis and 20 healthy volunteers during fasting and 30 min after ingestion of a standardized meal. Hemodynamic parameters were related to the degree of esophageal varices, variceal bleeding, portal hypertensive gastropathy and Child-Pugh score. RESULTS The postprandial portal blood velocity increment was low in patients with esophageal varices of any degree (22-24%), compared to patients without varices (49%, p<0.01) and healthy controls (65%, p<0.001), but was not different in patients with or without variceal bleeding (22% vs. 20%). In contrast, the congestion index (CI; ratio of portal vein cross-sectional area and portal blood velocity) pre-/postprandial decreased in the bleeding group only (CI pre/ CI post 1.30+/-0.23 (no bleeding) vs. 0.86+/-0.29 (bleeding); p<0.01). Portal hypertensive gastropathy was not related to any of the portal flow parameters. The portal blood velocity increment was comparable in controls (65%) and patients with Child-Pugh class A cirrhosis (56%), but lower in patients with class B (32%) and class C cirrhosis (15%, p<0.05 vs. class A). Also, there was no postprandial decrease in congestion index in patients with the most severe cirrhosis (p<0.01 class C vs. class A and B). CONCLUSIONS The postprandial rise in portal flow is inversely related to the severity of portal hypertension and liver cirrhosis, and may be a valuable parameter with respect to the risk of variceal bleeding.

Evidence for central nervous effects of corticotropin-releasing hormone on gastric acid secretion in humans.

In animals, corticotropin-releasing hormone (CRH) has been shown to decrease gastric acid secretion after intracerebral administration. Evidence exists that in man peptides have a direct access to the brain upon intranasal administration. This study aimed at assessing brain-mediated effects of CRH on gastric pH after intranasal administration in humans. Eleven healthy men were tested on 2 occasions in a double-blind within-subject cross-over comparison during treatment with CRH (versus placebo) administered intranasally at a dose of 20 micrograms every 10 min. Gastric pH values were measured continuously by a gastral pH tube. After 2 h of intranasal treatment, 6 micrograms/kg pentagastrin was injected subcutaneously. The subjects mood was assessed by an adjective list (EWL-N) at the end of each experimental condition. Intranasal CRH increased pH baseline values from (mean +/- SE) 1.70 +/- 0.31 to 2.62 +/- 0.53 (corresponding to 54%), whereas during intranasal treatment with placebo pH values remained unchanged (p < 0.05). After injection of pentagastrin, pH values decreased to 0.73 +/- 0.04 during placebo and to 0.93 +/- 0.14 during CRH treatment (n.s.). During treatment with CRH, subjects felt less tired (p < 0.05) and deactivated (p < 0.05). Plasma cortisol and CRH levels were not affected by intranasal CRH, excluding mediation of the CRH effects via resorption into the bloodstream, and TSH levels were slightly decreased by the end of the treatment. Results confirm the notion of a pathway for CRH from the nose to the brain, initiating, via central nervous mechanisms, inhibition of gastric acid secretion and a change of mood in humans.

Limited Heat-Shock Protein 72 Induction in Caco-2 Cells by L-Glutamine

Background/Aims:L-Glutamine (L-gln) is a conditionally essential amino acid which is consumed by certain tissues like the intestine in large amounts as energy source during critical illness. Apart from nutritive action, recent data suggest a link to heat-shock protein (hsp) induction. We investigated the effect of L-gln on hsp72 expression in the intestinal cell line Caco-2 under basal and heat-shock conditions and compared it with related amino acids. Methods: Total cellular protein was extracted and separated by SDS-PAGE. Immunoblots were performed with anti-hsp72 followed by chemiluminescence detection and densitometric scanning. Results: Following heat shock, hsp72 protein expression increased by 72 and 53% at 2 and 4 mmol/l L-gln, respectively, compared to heat shock alone (p < 0.05). Under basal conditions a limited increase occurred only at 8 mmol/l L-gln (p < 0.01). Levels remained unaffected when related amino acids including alanine, glutamate or glycine were supplemented under basal and heat-shock conditions. Similarly, the nonmetabolizable glutamine analogue DON or the toxic metabolite L-pyroglutamate did not induce hsp72. Conclusion: We conclude that the glutamine-mediated effect may be specifically attributed to the metabolic action of L-gln.

Clathrin-mediated endocytosis of high density lipoprotein3 in human intestinal Caco-2 cells. A post-embedding immunocytochemical study

The mechanism by which high density lipoprotein (HDL) removes excess cholesterol from intracellular sites has been the subject of much controversy. There is some evidence that HDL binds to specific cell surface receptors without internalization. Other evidence suggests that HDL is taken up by endocytosis, enters a pathway of endosomal trafficking and is resecreted from the cells (retroendocytsosis). In the present study, we investigated the distribution of apolipoprotein AI, the major protein constituent of HDL, in cultured intestinal Caco-2 cells employing post-embedding immunocytochemistry on LR White-embedded material. Cells grown under control conditions showed label for apolipoprotein AI in the endoplasmic reticulum. After incubation with native apolipoprotein E-free high density lipoprotein3 (HDL3) additional label for apolipoprotein AI was found in endosomes. These endosomes were observed near lipid droplets and in the basolateral cytoplasm. Further, it was demonstrated that label for apolipoprotein AI was colocalized with label for clathrin on the basolateral membrane. Our results support the concept that HDL3 is internalized and subsequently processed in an endosomal pathway in Caco-2 cells besides de novo synthesis of apolipoprotein AI.

Expression of liver plasma membrane transporters in gallstone-susceptible and gallstone-resistant mice.

We tested the hypothesis that differential expression of liver plasma membrane transporters might account for variations in biliary lipid secretion rates between gallstone-susceptible C57L/J and gallstone-resistant AKR/J mice. Plasma membrane fractions and total RNA isolated from livers of mice fed with a control or lithogenic (15% fat/1.25% cholesterol/0.5% cholic acid) diet were used for measurements of steady-state gene expression of hepatobiliary transport systems for bile salts (Ntcp1/Slc10a1, Oatp1/Slc21a1 and Bsep/Abcb11), phospholipids (Mdr2/Abcb4), organic anions (Mrp2/Abcc2) and organic cations (Oct1/Slc22a1). Irrespective of the diet, the steady-state gene expression of hepatobiliary transporters did not differ significantly between the two strains. Despite a higher basal bile flow and bile-salt secretion in C57L mice, Mrp2 (Abcc2) and Bsep (Abcb11) expression did not differ between the two strains. Elevated biliary phospholipid secretion in response to the lithogenic diet was linked to increased Mdr2 (Abcb4) protein expression, whereas the induction of Oct1 (Slc22a1) might reflect an enhanced uptake of choline for augmented phospholipid synthesis. In response to the lithogenic diet, Bsep (Abcb11) protein expression was up-regulated only marginally and bile salt secretion did not increase. The down-regulation of Ntcp1 (Slc10a1) protein expression might protect hepatocytes from high intracellular bile-salt loads. We conclude that variations in protein function rather than in the gene expression of liver plasma membrane transporters might account for variations in biliary lipid secretion rates. Our findings support the concept that the formation of lithogenic bile is caused by the hypersecretion of bile salts as a result of augmented availability of canalicular membrane cholesterol, possibly amplified by bile-salt-phospholipid uncoupling due to the increased bile flow.

Mevinolin, a competitive inhibitor of hydroxymethylglutaryl coenzyme A reductase, suppresses enterocyte esterification of exogenous but not endogenous cholesterol

Mevinolin (lovastatin), a competitive inhibitor of hydroxymethylglutaryl-coenzyme A reductase, directly inhibited acyl-CoA cholesteryl acyltransferase in rabbit intestinal microsomes at a dose of 20 micrograms/ml or more. Lineweaver-Burk analysis showed a competitive type of inhibition with respect to oleoyl-CoA. In cultured intestinal Caco-2 cells, mevinolin reduced [14C]oleate incorporation into cholesteryl-esters by 86% of controls at doses as low as 0.1 micrograms/ml. However, in cells whose activity of acyl-CoA cholesteryl acyltransferase was stimulated 7-fold by 10 mM mevalonolactone, a significant inhibitory effect on cholesteryl-ester formation could not be detected, even at 40 micrograms/ml of mevinolin. In contrast, cells supplied with liposomal cholesterol or cholesterol derived from low-density lipoproteins showed a marked reduction of cholesteryl-ester formation in the presence of 10 or 0.1 micrograms/ml of mevinolin, respectively. It is concluded that the observed suppressive effects of mevinolin on cholesterol esterification in cultured Caco-2 cells are indirect and possibly caused by changes in the acyl-CoA cholesteryl acyltransferase substrate pool or intracellular cholesterol transport.

Reduced cholesterol esterification in CaCo-2 cells by indirect action of pravastatin

In microsomal preparations of CaCo-2 cells pravastatin decreased cholesterol esterifying activity at 25 micrograms/ml to 82.5% and at 800 micrograms/ml to 56.2% of controls. Pravastatin reduced cholesteryl ester formation dose-dependently also in viable CaCo-2 cells. However, the maximal inhibition was by 90.4% at pravastatin concentration of 25 micrograms/ml, half maximal inhibition occurred between concentrations of 5 and 10 micrograms/ml. Addition of mevalonolactone, which serves as endogenous source of cholesterol, antagonized this effect. At 10 mM mevalonolactone (MVL) even doses up to 200 micrograms/ml of pravastatin were ineffective. On the other hand, pravastatin suppressed cholesteryl ester formation when acyl-CoA cholesterol acyltransferase (ACAT) (E.C. 2.3.1.26) activity was stimulated by addition of exogenous liposomal or Low Density Lipoprotein (LDL)-derived cholesterol. This inhibition was refractory to increasing amounts of exogenous cholesterol up to 400 micrograms/ml. Therefore we conclude that only excessive doses of pravastatin suppress ACAT activity directly. In viable cells the observed inhibition of cholesteryl ester formation is due to the block in de novo synthesis of cholesterol, causing a lack of substrate for ACAT and of non-sterol products of mevalonic acid. Furthermore pravastatin interferes with the esterification and/or intracellular transport only of exogenous cholesterol, confirming former results of a compartmentalized cholesterol metabolism in the enterocyte.

Intracellular Transport of High-Density Lipoprotein 3 in Intestinal Epithelial Cells (Caco-2) Is Tubulin Associated

Background: A retroendocytotic pathway for high-density lipoprotein 3 (HDL3) in cultured intestinal epithelial cell lines has been described. In small intestinal crypt cells and Caco-2, HDL3 is internalized, transported to lipid droplets and, after solubilization of these lipid droplets, resecreted. In the present study we examined the mechanisms of intracellular transport of HDL3 in the Caco-2 cell line. Methods: Apolipoprotein E free HDL3 was gold-labeled for transmission electron microscopy and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine iodide [DiI(3)] labeled for fluorescence and confocal laser scanning microscopy. For tubulin desintegration Caco-2 cells were incubated with taxol, colchicine and β- and γ-lumicolchicine. Tubulin staining was performed using a FITC labeled antibody. Uptake of HDL3 was quantified by FACS analysis. Results: HDL3 was rapidly internalized and found to be in contact with lipid droplets in the perinuclear region after 10 min. By transmission electron microscopy a frequent colocalization of HDL3-containing vesicles and tubular structures was demonstrated. The close association of HDL3-containing vesicles with fluorescence stained tubulin could be confirmed by confocal laser scanning microscopy. Preincubation of the cells with taxol and colchicine did not completely prevent internalization but reduced it during a 2-hour incubation period to less than 50% of the control cells. The transport of DiI(3)-labeled HDL3 to the lipid droplets in the perinuclear region was almost completely blocked by taxol and colchicine. Conclusion: Internalization and intracellular transport of HDL3 in intestinal epithelial cells (Caco-2) is dependent on a tubulin-mediated mechanism.