Lipopolysaccharide binding protein, interleukin-6 and C-reactive protein in acute gastrointestinal infections: value as biomarkers to reduce unnecessary antibiotic therapy.

AIM: Several new biomarkers, such as lipopolysaccharide binding protein (LBP) and interleukin-6 (IL-6), have the potential to determine the severity and outcome of infectious diseases. LBP and IL-6 serum levels have not been reported in patients with gastrointestinal infections. The aim of this study was to compare established markers of infection with new markers, such as LBP and IL-6, in patients with acute gastrointestinal infections METHOD: LBP, C-reactive protein (CRP), white blood cell count (WBC) and IL-6 serum levels were determined in patients with acute viral or bacterial (positive stool cultures) gastroenteritis. The final diagnosis and empiric antibiotic use were recorded. In total, medical data on 88 patients with acute gastroenteritis (22 bacterial, 66 viral or nonspecific) were analyzed. RESULTS: LBP and CRP levels were significantly increased in patients with acute bacterial gastroenteritis [28.5 ± 16.5 vs. 15.2 ± 11.5 µg/mL (p < 0.05) and 10.4 ± 9.6 vs. 3.8 ± 5.5 mg/dL (p < 0.001), respectively]. LBP at a cut-off value of 14.6 µg/mL and CRP at a cut-off value of 1.7 mg/dL distinguished between bacterial and non-bacterial gastrointestinal infection (receiver operator characteristic analysis). Empiric antibiotic therapy was initiated in 82% of patients with bacterial gastroenteritis and in 27% of patients with viral gastroenteritis. CONCLUSION: The use of the cut-off values for LBP and CRP determined here would have avoided unnecessary antibiotic therapy in 14 and 11%, of patients respectively. CRP and LBP appear to be superior to IL-6 and WBC as diagnostic markers of bacterial gastrointestinal infection. Cut-off values may be a useful tool to support clinical decision-making on whether or not to initiate empiric antibiotic therapy.

Mechanisms of hypotonic inhibition of the sodium, proton exchanger type 1 (NHE1) in a biliary epithelial cell line (Mz-Cha-1).

AIM: To elucidate the cellular events that results in inhibition of Na(+), H(+) exchanger type 1 (NHE1) by hypotonicity. METHODS: Intracellular pH (pH(i)) was measured in biliary epithelial cells, with the pH-sensitive fluorochrome 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) using a spectrophotometer. Regulatory volume decrease (RVD) was analysed from confocal images. Changes in NHE1 membrane content were visualized by confocal laser scanning microscopy after transfection of Mz-Cha-1 cells with a NHE1-cMyc fusion protein. RESULTS: In Mz-Cha-1 cells hypotonicity (-80 mmol L(-1) NaCl) inhibited endogenous Na(+), H(+) exchange. Tyrosine and serine kinase inhibitors were incapable to prevent inhibition. As several signalling pathways influence Na(+), H(+) exchange, we tested the effect of the Ca(++), Calmodulin, protein kinase C or the cAMP, protein kinase A system on inhibition of Na(+), H(+) exchange by hypotonic challenge, but neither system was involved. In contrast, cytoskeleton did influence the effect of hypotonicity. Inhibition of microtubule polymerization by colchicine prevented inhibition of NHE1, and also restored Na(+), H(+) exchange kinetics. Specific inhibition of Src kinases with PP2, attenuated pH(i) recovery rate from 1.93 +/- 0.16 pH units min(-1) (normotonic environment) to 1.02 +/- 0.50 pH units min(-1) (hypotonic environment). Membrane staining of NHE1-cMyc fusion protein was maintained after hypotonic exposure in colchicine pre-treated cells as was RVD. Microfilament inhibition by cytochalasin preserved NHE1 activity. Inhibition of phosphatidylinositol-3'-kinase was unable to restore Na(+), H(+) exchange activity. CONCLUSION: We conclude that regulation of Na(+), H(+) exchange during RVD is mediated by cytoskeletal elements. This receptor independent pathway is regulated by Src.

Budesonide for the treatment of obstructive eosinophilic jejunitis.

Eosinophilic gastroenteritis is a rare gastrointestinal (GI) disorder of undetermined origin, characterized by infiltration of eosinophils in the GI tract. Different layers of the bowel wall can be involved and the clinical outlook depends on the area affected. Our subject is a male patient in whom the disease involves the muscular layer causing obstructive jejunitis. The diagnosis was made after surgical resection. A relapse was subsequently treated with short-term intravenous steroids followed by oral budesonide for three months. Treatment was effective with no apparent side effects.

Progressive sclerosing cholangitis after septic shock: a new variant of vanishing bile duct disorders.

BACKGROUND: We present nine patients with progressive sclerosing cholangitis after septic shock. PATIENTS: All nine patients had previously required long term treatment in an intensive care unit for septic shock: two patients with polytrauma, five with burn injury, and two with extensive surgery. They were admitted to our hospital because of cholangitis. Endoscopic retrograde cholangiography revealed severe intrahepatic stenoses in all patients and liver biopsies showed typical signs of sclerosing cholangitis. No patient had pre-existing liver disease. RESULTS: Mean follow up time was 35 months. In patients with major bile duct stenoses (3/9), 12 endoscopic dilations were performed in total. In one patient, concrements were extracted and intermittent stenting was necessary. To date, 4/9 patients have rapidly developed liver cirrhosis. During follow up, 5/9 patients died: two after fulminant cholangitis, one after liver failure, one due to liver transplantation associated problems, and one after cerebral ischaemia. One patient has been registered for transplantation and the remaining three patients show no acute signs of liver failure. CONCLUSIONS: Patients with sclerosing cholangitis, following septic shock, represent a new variant of vanishing bile duct disorders. In such patients liver disease rapidly progresses to cirrhosis. Endoscopic treatment may only transiently improve the course of the disease. Orthotopic liver transplantation is indicated in end stage disease.

Procalcitonin is a valid marker of infection in decompensated cirrhosis.

BACKGROUND/AIMS: Bacterial infections are life-threatening complications in cirrhosis and early diagnosis is mandatory. Procalcitonin, a 116 amino acid propeptide of calcitonin, is an early marker of infection. The aim was to evaluate prospectively procalcitonin in the diagnosis of bacterial infection in cirrhosis. 127 patients with liver cirrhosis were analysed and stratified into three groups according bacteriological and morphological findings; decompensated patients with (group I = 36) and without (group II = 64) infection, and 27 non-decompensated and non-infected (group III). METHODS: Diagnosis of infection was made using standard criteria. Serum procalcitonin, tumour necrosis factor alpha, interleukin-6 and C-reactive protein were measured using commercially available methods. RESULTS: PCT serum levels were significantly different between group I (2.8 ng/ml [0.4 - 20.4]), group II (0.6 ng/ml [0.1 - 5.9]) and group III (0.4 ng/ml [0.1 - 1.2]), respectively. Levels above 0.58 ng/ml had a sensitivity of 92 % and specificity of 78 % for the diagnosis of infection and were associated with a 50 % mortality in the first two months. Interleukin-6, tumour necrosis factor alpha and C-reactive protein were less sensitive and specific for the diagnosis of infection. CONCLUSION: In decompensated cirrhosis procalcitonin serum levels provided the most sensitive and specific tool for the initial diagnosis of bacterial infection.

[MRI in chronic inflammatory bowel disease]. / MRT chronisch entzündlicher Darmerkrankungen.

Chronic inflammatory bowel disease is diagnosed and monitored by the combination of colonoscopy and small bowel enteroklysis. Magnetic resonance imaging has become the gold standard for the imaging of perirectal and pelvic fistulas. With the advent of ultrafast MRI small and large bowel imaging has become highly attractive and is being advocated more and more in the diagnostic work up of inflammatory bowel disease. Imaging protocols include fast T1-weighted gradient echo and T2-weighted TSE sequences and oral or rectal bowel distension. Furthermore, dedicated imaging protocols are based on breath-hold imaging under pharmacological bowel paralysis and gastrointestinal MR contrast agents (Hydro-MRI). High diagnostic accuracy can be achieved in Crohn's disease with special reference to the pattern of disease, depth of inflammation, mesenteric reaction, sinus tract depiction and formation of abscess. In ulcerative colitis, the mucosa-related inflammation causes significantly less bowel wall thickening compared to Crohn's disease. Therefore with MRI, the extent of inflammatory changes is always underestimated compared to colonoscopy. According to our experience in more than 200 patients as well as the results in other centers, Hydro-MRI possesses the potential to replace enteroklysis in the diagnosis of chronic inflammatory bowel disease and most of the follow-up colonoscopies in Crohn's disease. Further technical improvements in 3D imaging will allow interactive postprocessing of the MR data.

Endothelin-1 and smooth muscle cells: induction of jun amino-terminal kinase through an oxygen radical-sensitive mechanism.

Endothelin-1 (ET-1) has been proposed to contribute to atherogenesis and plaque rupture in coronary heart disease through activation of mitogen-activated protein kinases (MAPKs) in smooth muscle cells (SMCs). Reactive oxygen species (ROS) have been shown to be important signal transduction molecules in SMCs. Thus, the present study aimed to assess the role of ROS in ET-1-mediated activation of c-Jun amino-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2. Rat SMCs were exposed to ET-1 over time at concentrations from 10(-6) to 10(-10) mol/L, and MAPK activity was quantified. Activation of JNK and ERK was observed with a maximum stimulation at 10(-7) mol/L ET-1. JNK and ERK were activated by ET-1 binding to a single receptor (ET-1A) but differed in their downstream mechanisms: only JNK activation was sensitive to the radical scavenger N-acetylcysteine and diphenylene iodonium, an inhibitor of NADPH oxidase, indicating a role for ROS. The downstream MAPK effector and proinflammatory transcription factor, the activator protein-1 complex, was maximally activated 2 hours after the addition of ET-1. It was mainly composed of the JNK substrate c-Jun, and activation was also dependent on ROS formation. We suggest that plaque activation by ET-1 can be mediated through ROS. It can be hypothesized that the clinical benefit of antioxidants in the treatment of atherogenesis may partially depend on neutralization of ET-1-mediated ROS production.

Differential activation of mitogen-activated protein kinases in smooth muscle cells by angiotensin II: involvement of p22phox and reactive oxygen species.

The atherogenic effect of the renin-angiotensin system can be explained, in part, by the influence of its effector, angiotensin II (Ang II), on vascular smooth muscle cell (VSMC) growth. There is evidence that reactive oxygen species (ROS) play a role in the atherogenesis and activation of mitogen-activating protein (MAP) kinases, which are involved in proliferation and differentiation. The study was performed to further characterize the role of ROS in Ang II-mediated MAP kinase activation and the regulation of the transcription factor activator protein-1 (AP-1). Rat VSMCs were stimulated with Ang II. The activities of MAP kinases were assessed by Western blot analysis or by immunocomplex kinase assay. AP-1 binding was determined by using an electrophoretic mobility shift assay. Rat VSMCs were treated with Ang II-activated MAP kinases, extracellular signal-regulated kinase (ERK), c-Jun amino terminal kinase (JNK), p38 MAP kinase (p38 MAPK), and their downstream effector, AP-1. Interestingly, only the activation of ERK1/2, but not JNK or p38 MAPK, was tyrosine kinase, protein kinase C, and MEK1/2 dependent. Ang II also induced the rapid formation of ROS, which could be inhibited by a specific antibody as well as by antisense against the p22phox subunit of the NAD(P)H oxidase. JNK and p38 MAPK, but not ERK, activation was inhibited by an inhibitor of NAD(P)H oxidase. Antisense against p22phox also solely inhibited p38 MAPK but did not affect ERK. The results indicate that in VSMCs, Ang II activates MAP kinases and AP-1 through different pathways; the results further suggest that ROS, generated by p22phox, mediate Ang II-induced JNK and p38 MAPK activation, which may contribute to the pathogenesis of atherosclerosis.

Sodium, hydrogen exchange type 1 and bile ductular secretory activity in the guinea pig.

Biliary epithelial cells (BECs) express different Na(+), H(+) exchange (NHE) isoforms. In this study, the potential role of NHE in ductular bile secretion is assessed. Experiments were performed in guinea pig perfused livers and isolated BECs. Inhibition of NHE was achieved by hypotonic stress and by using the unspecific NHE inhibitor, amiloride, or the specific NHE 1 inhibitor, cariporide (HOE 642). Hypotonic stress inhibited basal bile flow by 46% and prevented secretin stimulation of bile flow by reducing biliary bicarbonate output by 50%. Secretin increased bile flow from 3.7 +/- 0.8 microL/min/g to 4.78 microL/min/g (P <.01); subsequent exposure to hypotonic stress decreased secretin-stimulated bile flow by 35% and biliary bicarbonate secretion by approximately 50%. Inhibition of NHE by amiloride or cariporide resulted in a similar reduction of secretin-stimulated bile flow and bicarbonate secretion. Basal bile flow was unaffected by the NHE inhibitors. In isolated guinea pig BECs, regulatory volume decrease and inhibition of NHE was demonstrated after hypotonic stress under basal and secretin-stimulated conditions. In contrast, hypotonic exposure inhibited Cl(-), HCO(3)(-) exchange activity in isolated BECs only during basal conditions but incompletely after secretin stimulation. Our study shows that hypotonic stress inhibits basal bile flow in the guinea pig by inhibition of Cl(-), HCO(3)(-) exchange. NHE1 is not involved in basal bile formation. Increased choleresis after ductular stimulation by secretin depends on intact NHE1 activity. These data indicate that BEC volume changes have profound effects on biliary secretory function.

Expression of a bile acid transporter in biliary epithelial cells from normal and cholestatic rat livers.

A sodium dependent bile acid carrier has recently been cloned and characterized in rat ileum. The present study demonstrates the presence of a mRNA species specific for the rat ileal bile acid carrier (r-IBAT) in rat biliary epithelial cells. Moreover, immunohistochemistry with a peptide specific antibody demonstrates protein expression in biliary epithelial cells from normal and bile duct ligated rat livers. Besides a cytoplasmic staining a predominant staining of the apical membrane could be observed. These observations indicate that biliary epithelial cells are involved in bile acid transport across the biliary tree. In addition the carrier could also play a role in the signal transduction of bile acid induced ductular secretion.

Electrogenicity of hepatocellular fatty acid uptake.

Sensitivity of cellular fatty acids uptake to the membrane potential difference is still a matter of controversy. For direct evaluation of potential sensitivity the effect of changing membrane potential on uptake of a fluorescent long chain fatty acid derivative, 12-NBD-stearate, in isolated rat hepatocytes, was examined. Changes in membrane potential were achieved by patch clamp procedures. Fatty acid influx was simultaneously determined by recording of cell fluorescence. Hyperpolarization from -30 to -70 mV accelerated fatty acid influx whereas depolarization to +50 mV reduced uptake. After obtaining equilibrium hyperpolarization increased cell fluorescence, whereas depolarization pushed NBD-stearate out of cells. Potential sensitivity of uptake was dependent on the fatty acid concentrations in the medium with most prominent effects at low unbound concentrations. These data show that, at low fatty acid concentrations, uptake is, in part, driven by an intracellular negative electric membrane potential.

Long-chain fatty acid uptake by skeletal myocytes: a confocal laser scanning microscopy study.

Studies of regulation of free fatty acid (FFA) utilization by skeletal muscles have focused on plasma FFA delivery and on intracellular factors affecting FFA metabolism. The present study was conducted to directly analyse the uptake process of fatty acids into single myocytes. Cells were isolated from the rat flexor digitorum brevis muscle. Confocal laser scanning microscopy was utilized to analyse the uptake of the fluorescent fatty acid derivative 12-NBD-stearate, which is not metabolized by muscle tissue. Uptake represented a saturable function of the unbound fatty acid concentration in the medium (K(m) 366 +/- 118 nM, Vmax 2.1 +/- 0.3 AU/s) and depended on the medium sodium concentration. Reduced buffer pH increased initial uptake rates, whereas lactate (10 mM) had no effect. Membrane hyper- and depolarization decreased uptake rates. This study demonstrates for the first time kinetic data from isolated myocytes with evidence for a carrier-mediated transport mechanism for long-chain fatty acids.

Muscarinic acetylcholine receptor stimulation of biliary epithelial cells and its effect on bile secretion in the isolated perfused liver [corrected].

The aim was to explore whether biliary epithelial cells show muscarinic acetylcholine receptors and to investigate their role in ductular bile formation. In both, isolated rat biliary epithelial cells and Mz-Cha-1 cells, a biliary epithelial cell line, binding of [3H]N-methyl-scopolamine occurred with 0.718 +/- 0.08 and 0.482 +/- 0.05 fmol per 10(6) cells, respectively. To characterize the involved second messenger, intracellular Ca2+ levels were monitored by confocal microscopy. Stimulation of biliary epithelial cells with carbachol produced an increase in free cytosolic Ca2+ levels that declined to baseline values describing a sinusoidal oscillation curve. Increasing concentrations of the agonist decreased latency of the response and increased oscillation frequency. Similar results were obtained in Mz-Cha-1 cells. The intracellular Ca2+ originated from IP3 sensitive intracellular stores and from the extracellular medium. The Ca2+ response could partially be blocked by atropine and completely by pirenzepine, a specific muscarinic receptor-type M1 antagonist. The presence of M1 receptor messenger RNA (mRNA) in biliary epithelial cells was confirmed by reverse transcriptase polymerase chain reaction. In the isolated perfused guinea pig liver, a model with high ductular bile flow, carbachol induced a dose dependent decrease of bile flow by 79.6% +/- 9.8% at 50 mumol/L carbachol (P < .001), without affecting perfusion pressure or biliary electrolyte concentrations. It is concluded that biliary epithelial cells express muscarinic acetylcholine receptors. Stimulation of this receptor leads to cholestasis. This could be because of changes in peribiliary permeability and/or inhibition of biliary epithelial cell secretory function.

Effect of ethanol and fructose on liver metabolism: a dynamic 31Phosphorus magnetic resonance spectroscopy study in normal volunteers.

In vivo 31Phosphorus magnetic resonance spectroscopy (31P-MRS) permits evaluation of dynamic changes of individual phosphorus-containing metabolites in the liver parenchyma, such as phosphomonoester (PME), adenosine triphosphate, and inorganic phosphate (Pi). Intravenous fructose load alters phosphorus metabolites and allows assessment of liver function by 31P-MRS. 31P-MRS data obtained in alcoholic liver disease are however inconclusive. To study the hypothesis that fructose load can be used to investigate metabolic effects of ethanol ingestion, the interaction of different metabolites--i.e., fructose and ethanol--were followed in vivo. Using a 1.5 Tesla magnetic resonance system, six healthy volunteers were examined in three sessions each: a session after administration of (a) fructose only (250 mg/kg) was compared with (b) fructose load after ethanol ingestion (0.8 g/kg). A control experiment (c) was done after ethanol only. Spectra were acquired using one-dimensional chemical shift imaging with a temporal resolution of 5 min. Following a fructose load, the concomitant uptake of ethanol showed drastic changes of individual metabolic steps of the hepatic metabolism (averages +/- standard deviation). While the velocity of the net formation of PME (relative increase 0.46 +/- 0.11 without ethanol vs. 0.61 +/- 0.25 with ethanol) and the use of adenosine triphosphate (-0.13 +/- 0.03 vs. -0.16 +/- 0.03) and Pi (-0.022 +/- 0.009 vs. -0.021 +/- 0.004) were not significantly affected by ethanol uptake, a significant (p < 0.01) reduction of PME degradation (31.3 +/- 9.4 vs. 61.9 +/- 16.9 relative total area) and absence of an overshoot for Pi (10.5 +/- 4.9 vs. -7.1 +/- 5.3 relative area 13 min to 43 min) was observed after ethanol administration. Dynamic 31P-MRS allows the observation of individual steps of hepatic metabolism in situ; fructose metabolism in the human liver is slowed down by concomitant ethanol ingestion after the phosphorylation step of fructose. This could be explained by inhibition of aldolase rather than ethanol-induced changes of the hepatic redox state. Fructose load can be used to study effects of alcohol ingestion and might therefore be useful in patients with alcoholic liver disease.

Effect of surface and intracellular pH on hepatocellular fatty acid uptake.

Fatty acids enter hepatocytes, at least in part, by a carrier-mediated uptake mechanism. The importance of driving forces for fatty acid uptake is still controversial. To evaluate possible driving mechanisms for fatty acid transport across plasma membranes, we examined the role of transmembrane proton gradients on fatty acid influx in primary cultured rat hepatocytes. After hepatocytes were loaded with SNARF-1 acetoxymethyl ester, changes in intracellular pH (pHi) under different experimental conditions were measured and recorded by confocal laser scanning microscopy. Fatty acid transport was increased by 45% during cellular alkalosis, achieved by adding 20 mM NH4Cl to the medium, and a concomitant paracellular acidification was observed. Fatty acid uptake was decreased by 30% during cellular acidosis after withdrawal of NH4Cl from the medium. Cellular acidosis activates the Na+/H+ antiporter to export excessive protons to the outer cell surface. Inhibition of Na+/H+ antiporter activity by amiloride diminishes pHi recovery and thereby accumulation of protons at the outer surface of the plasma membrane. Under these conditions, fatty acid uptake was further inhibited by 57% of control conditions. This suggests stimulation of fatty acid influx by an inwardly directed proton gradient. The accelerating effect of protons at the outer surface of the plasma membrane was confirmed by studies in which pH of the medium was varied at constant pHi. Significantly higher fatty acid influx rates were observed at low buffer pH. Recorded differences in fatty acid uptake appeared to be independent of changes in membrane potential, because BaCl2 did not influence initial uptake velocity during cellular alkalosis and paracellular acidosis. Moreover, addition of oleate-albumin mixtures to the NH4Cl incubation buffer did not change the observed intracellular alkalinization. In contrast, after cells were acid loaded, addition of oleate-albumin solutions to the recovery buffer increased pHi recovery rates from 0.21 +/- 0.02 to 0.36 +/- 0.05 pH units/min (P < 0.05), indicating that fatty acids further stimulate Na+/H+ antiporter activity during pHi recovery from an acid load. It is concluded that carrier-mediated uptake of fatty acids in hepatocytes follows an inwardly directed transmembrane proton gradient and is stimulated by the presence of H+ at the outer surface of the plasma membrane.

Sodium, hydrogen antiporter activation by extracellular adenosine triphosphate in biliary epithelial cells.

BACKGROUND & AIMS: Extracellular nucleotides are secretagogues and influence ion permeability. The aim of this study was to investigate whether secretagogues activate transmembrane acid-base carriers, e.g., sodium, hydrogen antiporter in cholangiocytes. METHODS: Cells were loaded with pH and Ca(2+)-sensitive dyes. Intracellular changes in ion concentrations were monitored by confocal laser scanning microscopy. Adenosine 3', 5'-cyclic monophosphate was determined by standard methods. RESULTS: Baseline intracellular pH (pH1) averaged 7.28 +/- 0.17 pH units. Adenosine triphosphate (ATP; 10 mumol/L) increased baseline pH1 by 0.28 +/- 0.06 pH units/10 min (P < 0.01). Ten and 100 mumol/L ATP increased Na+, H+ antiporter-mediated proton flux from 9.4 +/- 4.9 mmol. L-1 min-1 to 17.2 +/- 11.8 and 16.7 +/- 10.3 mmol. L(-1)-min(-1) (P < 0.001), respectively. Under control and ATP-stimulated conditions, 1 mmol/L amiloride blocked pH1 recovery, indicating true activation of Na+, H+ antiporter by extracellular ATP. Inhibition of basolateral Na+, H+ antiporter isoform inhibited stimulation by ATP. Na+, H+ antiporter-mediated proton flux was stimulated by adenosine, uridine triphosphate, adenosine-5'-0-(3-thiotriphosphate), alpha, beta-methylene-ATP, and 2-methylthio-ATP but not prevented by adenosine receptor blocking. Activation by ATP was not influenced by the Ca2+/protein kinase C/calmodulin system but could be mimicked by addition of N6,2'-O-dibutyryladenosine-3',5'-cyclic monophosphate and was inhibited by pertussis toxin. CONCLUSIONS: Extracellular nucleotides may modulate secretory and absorptive function of cholangiocytes by activating Na+/H+ exchange mechanisms.

Rapid onset of apoptosis in vitro follows disruption of beta 1-integrin/matrix interactions in human colonic crypt cells.

BACKGROUND & AIMS: Apoptosis by loss of adherence is a recently described phenomenon termed "anoikis." beta 1, integrins are heterodimeric surface molecules mediating adhesion to the extracellular matrix. The aim of this study was to address whether anoikis accounts for the elimination of senescent enterocytes and, if so, whether beta 1 integrins are involved. METHODS: Whole crypts were isolated from normal human colonic mucosa and examined in vitro. RESULTS: The vast majority of cells in resuspended crypts rapidly underwent apoptosis within 4 hours. Apoptosis was partially inhibited when cells had contact with collagen I-coated membranes or when whole crypts were embedded in a collagen gel. Preincubation of crypts with an inhibiting anti-beta 1 antibody before readhesion caused a much higher apoptotic rate. Confocal microscopy of embedded crypts revealed two critical zones of high sensitivity to temporary loss of adherence: the base of the crypts where stem cells are supposed to reside and the crypt mouth including the surface epithelium. CONCLUSIONS: Survival of colonic epithelia crucially depends on matrix adhesion and is likely to be guaranteed by beta 1-integrin/ matrix interaction. The data strongly suggest that anoikis is the way senescent colon cells die.

Differential expression of Na+,H(+)-antiporter mRNA in biliary epithelial cells and in hepatocytes.

BACKGROUND/AIMS: Amiloride inhibitable Na+,H(+)-exchange has recently been identified and characterized in rat biliary epithelial cells where its activity at low intracellular pH is significantly higher than in hepatocytes. METHODS: Northern blot and reverse transcription-polymerase chain reaction were used to study the expression of the different Na+,H(+)-antiporter isoforms in isolated biliary epithelial cells and hepatocytes. RESULTS: The present study demonstrates for the first time the expression of Na+,H(+)-antiporter isoform 1 mRNA in rat biliary epithelial cells. Moreover, steady-state levels of this message were several-fold higher in biliary epithelial cells than in hepatocytes. In addition, the expression of Na+,H(+)-antiporter isoform 2 in bile duct epithelial cell but not hepatocytes could be demonstrated by reverse transcription-polymerase chain reaction. CONCLUSIONS: The higher expression of Na+,H(+)-antiporter isoform 1 mRNA may indicate a higher rate of synthesis and therefore a higher Na+,H(+)-exchange activity in biliary epithelial cells than in hepatocytes and is entirely compatible with the results of the previous functional studies.