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Background Extracorporeal shock wave lithotripsy (ESWL) of pancreatic duct stones followed by ERCP with mechanical clearance of the pancreatic duct and subsequent stenting is an established treatment option for chronic calcific pancreatitis. Objective To test the efficacy of a modified transportable mini-lithotripter for ESWL of pancreatic duct stones. Design Prospective single-center study. Setting University hospital. Patients This study involved 32 patients with obstructive chronic calcific pancreatitis and pain in whom previous endoscopic stone removal and pancreatic duct decompression had failed. Interventions ESWL followed by ERCP for stone clearance of the pancreatic duct and mechanical removal of stones or stenting. Main Outcome Measurements Endoscopic duct clearance and/or stent insertion, pain and quality-of-life scores. Results A median of 4 ESWL sessions (interquartile range 2.75-8.5) with a median of 6800 shock waves (4225-15,425) were required. Pain relief after ESWL only was noted in 24 patients (75.0%), whereas no change in the intensity of pain was reported by 7 patients (21.9%), and pain was worse in 1 patient. All patients underwent ERCP and stent placement, resulting in complete resolution of pain in 17 patients (53.1%) and pain improvement in 28 patients (87.5%). The quality-of-life score was significantly improved after ESWL and endoscopic clearance or stenting in all patients. Limitations Uncontrolled study. Conclusions ESWL with the mini-lithotripter results in fragmentation of pancreatic duct calculi. ESWL in conjunction with endoscopic clearance of the pancreatic duct and stenting is associated with significant improvement in clinical outcome and quality of life in patients with obstructive calcific chronic pancreatitis.

The aim of this study is to compare the histological grading of acute organ rejection according to the Banff score with intracellular interleukin-2 (IL-2) concentrations in cytotoxic CD8+ T cells from peripheral blood samples. 66 recipients after liver transplantation and 20 healthy controls were included into this study. Blood samples of liver transplant recipients were collected beside routine visits or, in case of suspected organ rejection, with additional liver biopsy. For cytometry, the blood cells were stained with CD3, CD8 and intracellular-IL-2. The percentage of cells with detectable intracellular IL-2 was significantly increased in patients with acute rejection (n = 7, P0.001, t Test) compared to recipients without rejection. The percentage of cells with detectable intracellular IL-2 (mean +/- SEM) was 7.6 +/- 0.9% in rejection patients, 2.3 +/- 0.22% in stable liver transplant recipients, and 14 +/- 2.99% in healthy controls. Intracellular IL-2 correlates to the Banff score in rejection patients (Spearmans-rho = 0.81, P0.05). This cytometric method shows a good sensitivity (71%) with a cut-off based on a high specificity of 95% for histological proven organ rejection in our study cohort. Measurement of intracellular IL-2 in cytotoxic CD8+ T-lymphocytes by flow cytometry correlates very well to the histological grading according to the Banff score and shows a good sensitivity and excellent specificity in acute organ rejection.

Background. Amyloidosis represents a group of different diseases characterized by extracellular accumulation of pathologic fibrillar proteins in various tissues and organs. Severe amyloid deposition in the liver parenchyma has extrahepatic involvement predominantly in the kidney or heart. We evaluated the effect of ursodeoxycholic acid, in four patients with severe hepatic amyloidosis of different etiologies, who presented with increased alkaline phosphatase and ?-glutamyl transferase. Case report. The study included four patients who presented with amyloidosis-associated intrahepatic cholestasis. Three of them had renal amyloidosis which developed 1-3 years before cholestasis occurred, the remaining one having intrahepatic cholestasis as the primary sign of the disease. Amyloidosis was identified from liver biopsies in all patients by its specific binding to Congo red and green birefringence in polarized light. The biochemical nature and the class of amyloid deposits were identified immunohistochemically. In addition to their regular treatment, the patients received 750 mg ursodeoxycholic acid per day. After 2-4 weeks all patients had a significant decrease of serum alkaline phosphatase and ?-glutamyl transferase, and their general status significantly improved. Conclusion. Treatment with ursodeoxycholic acid may be beneficial in patients with hepatic amyloidosis, and do extend indications for the use of ursodeoxycholic acid in amyloidotic cholestatic liver disease.

Introduction. In the last two decades, an increase in obesity along with a number of co-morbidities has been recorded among children and adolescents. Objective. The aim was to investigate if there was a difference in nutritional status between adolescents who were active sportsmen and those who did not engage in sport activities other than regular physical activities at school and if active training could be an independent factor in the prevention of obesity. Methods. There were 117 male adolescents on average aged 15.4?1.8 years. The subjects were divided into two groups; 32 active sportsmen engaged in regular training in football and a control group consisting of 85 adolescents engaged in no physical activity except for that at school (two hours per week). In all subjects Body Mass Index (BMI) was determined. The percentage of the total body fat was estimated from the sum of values of skin fold thickness measured at four sites (according to Durnin and Womersley). Lean Body Mass (LBM) was estimated by deducting the triceps skin fold from the upper arm circumference and comparing it to the norms. All collected data were analyzed by descriptive and analytical statistical methods: ?2-test, t-test. Results. Adolescent sportsmen showed a statistically significantly lower BMI and percentage of total body fat than the adolescents in the control group (p

In this study we characterized a model of human peritoneal macrophages maintained in culture for up to 48 h that can be used to study different functions of this cell population in vitro. The cells remained viable and functionally active over time, with well-preserved phagocytic properties. They expressed a macrophage marker, CD14. Once in culture, human peritoneal macrophages secreted C1q and nitric oxide in a pattern described in murine, guinea pig, and rat peritoneal macrophages . The described model can be used to study physiology and pathophysiology of peritoneal macrophages in vitro, offering all the advantages of the use of a human cell population.

Background Deoxycholic acid has long been attributed as a tumour promoter in the colon. It exerts its growth-related actions in a phorbol ester-like manner, by stimulating protein kinase C. The aim of this study was to investigate the effect of deoxycholic acid on proliferation and apoptosis in the colon, by exposing colon cancer cells to it in increasing concentrations. Methods Human colon cancer cells (Caco-2 and HT-29) were treated with deoxycholate or its two structural isomers, 3-beta-12-alpha-dihydroxy-5-beta-cholan-24-oic acid and 3alpha-12-beta-dihydroxy-5-beta-cholan-24-oic acid. Proliferation was evaluated by cell counting, and apoptosis by estimating percentage cell survival and assessment of nuclear morphology. Results Within the concentration range of up to 20 aeM, deoxycholate stimulated growth of both human colon cancer cell lines. Its growth-promoting effect was abolished after inhibition of protein kinase C. At concentrations above 100 aeM, deoxycholate induced apoptosis in both cell lines. Epimers of deoxycholate were significantly less potent in stimulating growth. Conclusion Low-dose deoxycholate stimulates colon cancer cell proliferation while > 100 aemol L -1 of this secondary bile acid induces apoptosis in colon cancer cells. Deoxycholate might promote the likelihood of malignant transformation by increasing epithelial cell turnover in the colon.

Background Alpha1-proteinase inhibitor (alpha1-PI), an anti-inflammatory protein thought to play a role in the intestinal inflammation, is synthesised by and released from the intestinal epithelial cells. IL-1beta is a key proinflammatory cytokine in the abnormal immune response that occurs in inflammatory bowel disease. Butyrate is a normal luminal constituent in the colon, known to be of benefit in preventing inflammatory bowel disease. Direct modes of action of butyrate in intestinal inflammation have been poorly studied so far. The aim of this study was to investigate the effects of butyrate on cytokine-mediated alpha1-PI release in intestinal epithelial cells. Methods Differentiated Caco-2 cells were incubated with IL-1beta in the presence or absence of 2 mM butyrate. Alpha1-PI expression in the cells was evaluated by Western blot analysis and alpha1-PI release by ELISA. Results Treatment with butyrate alone had no effect on alpha1-PI expression in differentiated Caco-2 cells. However, treatment of the cells with 2 mM butyrate significantly reduced the alpha1-PI level in IL-1beta-treated cells. In the cell culture medium, the presence of butyrate impaired the IL-1beta-induced alpha1-PI release to 17-35%. The treatment induced no change in the number of detached cells or the percentage of viable cells. Conclusion Our data show that butyrate inhibits alpha1-PI release from Caco-2 colonocytes treated with IL-1beta. It is therefore likely that anti-inflammatory actions of butyrate occur via a mechanism that does not involve direct regulation of cytokine-induced anti-inflammatory protein expression in intestinal epithelial cells.

Background Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit colonic tumour-igenesis and have an established usefulness in cancer prevention. Because polyamines are essential for neoplastic cell growth, the aim of this study was to evaluate the effect of NSAIDs (indomethacin, a nonselective COX-1 and COX-2 inhibitor) on polyamine metabolism in colon cancer cells. Methods Both cell counting and thymidine incorporation into cellular DNA were used to assess colon cancer cell growth. Activities of polyamine-metabolising enzymes, polyamine content (HPLC) and ODC and c-myc protein expression (Western blot) were measured in colon cancer cells treated with indomethacin during logarithmic phase of proliferation. Results Indomethacin impaired growth of human colon cancer cells (Caco-2 and HCT-116). As a result, ornithine decarboxylase activity and c-myc protein expression were decreased. Treatment with indomethacin induced intracellular oxidant formation in colon cancer cells significantly increased the spermidine/spermine-acetyltrasferase activity (SSAT) and enhanced polyamine acetylation and efflux from colon cancer cells. Impairment of cell growth by indomethacin could not be reversed by exogenous polyamines. Conclusion Taken together, our results suggest that NSAIDs affect polyamine metabolism in colon cancer cells by inducing SSAT activity, and that polyamine depletion in NSAID-treated colon cancer cells is mainly due to enhanced polyamine acetylation and irreversible depletion of intracellular polyamine pools.

The aim of this study was to evaluate the effects of natural short-chain fatty acids (butyrate, propionate, valerate, acetate) and structural analogues of butyrate and propionate on cell growth and apoptosis in three human colonic adenocarcinoma cell lines (HT-29, Colo-320, and SW-948). We have previously shown that mercapto- and bromo-analogues of butyrate and propionate compete with natural short-chain fatty acids for uptake in the colonocyte. Among naturally occurring short-chain fatty acids, butyrate was the most potent inhibitor of proliferation in all three cell lines. Propionate exhibited a weaker antiproliferative effect, while other short-chain fatty acids (valerate, acetate) were ineffective. Bromo-analogues of butyrate and propionate were more potent proapoptotic agents than butyrate. In contrast to butyrate, the analogues induced strand breaks on isolated supercoiled DNA, the effect being completely reversed by a DNA-protecting agent, spermine. We conclude that bromo-analogues of butyrate and propionate are more potent proapoptotic agents than butyrate in colon cancer cells in culture. Their effect may be a result of direct DNA damage.

Deoxycholic acid (DCA) has long been implicated as tumour-promoting agent in the colon. Polyamines are necessary for cell proliferation, they are accumulated in high amounts in colon cancer cells, and their concentrations in the colonic lumen can reach millimolar levels. The aim of this study was to investigate the effects of physiological DCA concentrations on proliferation and polyamine content in human colon cancer cells (Caco-2) in culture. Over an initial 48 h in culture, DCA stimulated Caco-2 cell proliferation rate three-fold, reaching a maximum with 20 microM DCA. DCA-induced increases in ornithine decarboxylase (ODC) activity corresponded to peak proliferation rates, occurring only during the initial 48 h of cell proliferation. Treatment with low-dose DCA resulted in a two-fold increase in putrescine uptake, first noted after 2 days in culture, but persisting until the cells became confluent (day 5). Both basal and DCA-stimulated putrescine uptake in Caco-2 cells were saturable. Kinetic analysis of the uptake data showed that DCA-stimulated putrescine uptake was due to an increase in the capacity of the putative putrescine transporter, without changes in its affinity, therefore implying an increased number of putrescine transporters in the cell membrane, without change in their structure.

Background Physical exercise and testosterone administration result in a series of adaptive anabolic phenomena in the skeletal muscle. The role of polyamines in these processes has been poorly explored. Design We measured the activities of polyamine-synthesising enzymes, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) and polyamine content in skeletal muscle of male rats exposed to endurance or resistance exercise, or a single testosterone treatment. Soleus muscle (consisting mainly of slow-twitching oxidative fibres—STO) and extensor digitorum longus (mainly fast-twitching glycolytic muscle fibres—FTG) were analysed for polyamine content by HPLC, and ODC and SAMDC activity. Results Both endurance and resistance exercise induced a threefold increase in endogenous testosterone production. Two hours after exercise, ODC was increased in STO fibres, returning to baseline after 24 h; in FTG fibres the increase was less prominent. An increase in SAMDC activity occurred in a more sustained manner, with its peak 8 h after exercise. Polyamines were subsequently accumulated in both skeletal muscle fibres, with a rise in putrescine concentration after 2 h, and a fall corresponding to conversion of putrescine to spermidine and spermine by SAMDC. Single dose of 17α-methyltestosterone resulted in a similar increase in polyamine-synthesising enzyme activities and polyamine concentrations in the skeletal muscle. Conclusion Polyamine accumulation in the skeletal muscle after physical exercise is likely to occur secondary to testosterone production. Polyamines are apparently involved in the oxidative, but not in glycolytic processes related to muscle adaptation to exercise.

Background: Polyamines (putrescine, spermidine and spermine) are ubiquitous molecules indispensable for cell proliferation. In the intestinal lumen they are present in high amounts. Polyamine accumulation in proliferating cells of the intestinal mucosa is high, and it occurs both by enhanced synthesis and by increased uptake from the lumen. Aims: To study mitogen-induced polyamine accumulation in the gut, we treated proliferating Caco-2 cells with epidermal growth factor (EGF) and measured the activity of ornithine decarboxylase (ODC) and putrescine uptake. Furthermore, we investigated whether EGF-induced changes in the apical membrane could be responsible for the effect of EGF on polyamine uptake in Caco-2 cells. Methods: Putrescine uptake, ODC activity and intracellular polyamine content were evaluated in the presence of 100 ng/ml EGF. To study the mechanisms of EGF-stimulated polyamine uptake, apical membrane vesicles were isolated, and putrescine uptake into the vesicles measured. Possible enrichment in brush border membrane cytoskeleton proteins (ezrin and villin) was assessed by Western blot. Results: Treatment with EGF induced an increase in ODC activity, which occurred within the first minutes of treatment and reached peak values after 3 h. In contrast, an increase in putrescine uptake was more sustained, with peak levels at 12 h. Both synthesis and uptake contributed to an over 60% increase in intracellular putrescine and spermidine after EGF treatment. There were no detectable changes in apical membrane cytoskeleton (as concluded by the absence of ezrin and villin enrichment in EGF-treated Caco-2 cells). However, in apical membrane vesicles isolated from EGF-pretreated cells, putrescine uptake was enhanced twofold. Conclusions: EGF stimulates both synthesis and uptake of polyamines in Caco-2 cells. Enhanced synthesis seems to ensure rapid supply with polyamines in the earliest stages of growth, while the uptake is responsible for the maintenance of high polyamine intracellular levels during late growth phases. EGF-stimulated polyamine uptake is apparently not a consequence of structural changes in the apical membrane, but is likely to occur by a distinct EGF-induced alteration of the polyamine transporter itself.

We investigated the effects of epidermal growth factor (EGF) on polyamine uptake in Caco-2 cell monolayers. Cells were grown until confluence (day 7) or until differentiation (day 14). Polyamine uptake into Caco-2 cells was stimulated by EGF in a dose-dependent manner. Both basal and EGF-stimulated uptake rates were higher in 7- than in 14-day-old Caco-2 cells. Stimulation with EGF resulted in a significant increase in Vmax and an increased affinity for putrescine and spermine. Polyamine uptake was not inhibited when protein synthesis was blocked by cycloheximide, implying that no additional protein synthesis occured for stimulatory effect of EGF on polyamine uptake. The tyrosine kinase inhibitor, genistein, completely inhibited EGF-stimulated polyamine uptake, indicating that tyrosine phosphorylation plays a role in EGF-stimulated polyamine uptake in Caco-2 cells. The effect of EGF on polyamine uptake into Caco-2 cells, therefore, could be due to translocation of intracellular proteins which were not previously incorporated into the membrane, or direct alteration of polyamine transporter.

The application of panacyl bromide, a fluorescence labeling reagent for carboxylic acids, was examined. Fatty acids are separated by reversed-phase high-performance liquid chromatography. The derivatives of series of both saturated and unsaturated fatty acids (C10:0-C20:4) are simultaneously separated by a continuous gradient elution method using a methanol-based solvent containing acetonitrile. The quantitative detection is linear over a range of 10–500 pmol per injection. The method was demonstrated to be able to quantify free fatty acids in blood samples from normal subjects and diabetic patients.

Conditionally immortalized intestinal epithelial cells - A new model for studying intestinal epithelial cell turnover

There is an increasing evidence, stemming from epidemiological studies as well as studies performed in human biopsies and animal and cell culture models, suggesting that folate is chemopreventive in colonic carcinogenesis. Hyperhomocysteinemia is frequently associated with folate deficiency. Homocysteine, an amino acid, is metabolized to methionine in a 5-methyltetrahydrofolate (5-MTHF) dependent reaction.The aim of this study was i) to evaluate the effects of folate and its metabolites on growth and cell cycle progression in human colon cancer cells (Caco-2) in culture, and ii) to assess the effects of exogenous homocysteine on colon cancer cell proliferation. iii) Having found that homocysteine enhances colon cancer cell growth while metabolites of folate inhibit cell proliferation, we investigated the effects of simultaneous treatment in colon cancer cells.Caco-2 cells were incubated either with homocysteine (0.1-10 microM), and/or with folic acid and its metabolites (0.625-10 microg/ml). Cell proliferation was determined after 24 h and 48 h by measuring 5- bromo-2'-desoxyuridine (BrdU) incorporation. Additionally, the cells were trypsinized and prepared for cell cycle determination using propidium iodide for DNA staining. The stained cells were analyzed using a flow cytometer.Folate inhibited cell proliferation moderately within 24 h. Its metabolites, dihydrofolate and 5-MTHF were more potent inhibitors of cell growth. Treatment with folate and 5-MTHF resulted in the accumulation of cells in G1-G0 phase of the cell cycle and decreased the number of cells in G2-M phase. In addition, cells treated with 5-MTHF were predominantly accumulated in the S-phase. There was no difference in cell cycle progression of Caco-2 cells treated with homocysteine in comparison to controls. In homocysteine-treated cells, both folate and 5-MTHF reversed the homocysteine-induced enhancement of growth. In contrast, folate reduced the Caco-2 cell growth rate to control values and 5-MTHF depleted growth of homocysteine-treated cells to levels significantly lower than controls.Our data suggest that 5-MTHF, being the key metabolite in both the folate and homocysteine metabolic pathway, is the main modulator of growth-promoting actions of homocysteine as well as antiproliferative effects of folate in colon cancer cells.

We characterised a novel, charge-insufficient isosteric analogue of spermine, 11-[(amino)oxy]-4,9-diaza-1-aminoundecane (AOSPM). This analogue was synthesised by displacing aminopropyl group by aminooxyethyl group, the latter having pKa of about 5. Charge deficiency of the AOSPM molecule was fixed at a definite atom, while pKa of the rest nitrogen was similar to the parent polyamine. AOSPM competed with putrescine, spermidine and spermine for the uptake into the cell, and was accumulated in the cells in high amounts when exogenous polyamine synthesis was impaired. It was not recognised by the cells as growth-promoting polyamine, since it was unable to restore growth arrest due to polyamine deprivation. Like natural spermine, this polyamine analogue prevented oxidative DNA damage. AOSPM could be used not only as a tool to study polyamine homeostasis in the cell, but may have distinct applications either as radiation protector, a stable and non-toxic inhibitor of polyamine uptake or, as an appropriate vector, to enhance the uptake of impermeable compounds into the cell.

The complement system plays an important role in the humoral immune response. Activation of the classical complement pathway is mediated by its subcomponent, C1q, which is involved in the pathogenesis of several autoimmune disorders. Among the main C1q-synthesising tissues, macrophages have been attributed as the main source. We investigated the effects of anti-inflammatory drugs (methylprednisolone and acetylsalicylic acid (ASA)) on C1q secretion in human peritoneal macrophages in vitro. The macrophages were isolated from peritoneal lavage fluid of patients with end-stage renal disease undergoing continuous ambulatory peritoneal dialysis, and were maintained in culture for up to 6 days. ASA decreased while methylprednisolone increased C1q secretion from human peritoneal macrophages in vitro, which correlated well with the percentage of CD14 positive cells after treatment. We conclude that different response of the macrophages to treatment with methylprednisolone and ASA may point out to the importance of macrophage activation after treatment, as well as an increased abundance of membrane C1q accompanied by increased phagocytosis.

alpha1-Proteinase inhibitor (alpha1-PI) is the main serine proteinase inhibitor in human plasma. Apart from its synthesis in the liver, this anti-inflammatory protein is also synthesized by and excreted from human intestinal epithelial cells. Antiinflammatory actions of alpha1-PI are thought to be of relevance in the pathogenesis of inflammatory bowel disease. To investigate the role of macrophage-derived cytokines on alpha1-PI secretion from intestinal epithelial cells, we cultured Caco-2 cells until differentiation (14 days in culture) on permeable filter supports. Monolayers of differentiated Caco-2 cells were then co-cultured with human peritoneal macrophages, grown on plastic in the basolateral chamber. Under these conditions, alpha1-PI secretion from Caco-2 cells was enhanced by 45%, probably by a direct action of macrophage-derived cytokines on Caco-2 cells. To extend this observation further, we treated differentiated Caco-2 cells with macrophage-derived proinflammatory cytokines (IL-1beta, IL-8, TNF-alpha), as well as with lymphocyte-derived cytokines IL-2, IL-6 and IFN-gamma. As early as after 24h treatment, IL-2 and IL-8 induced a significant and dose-dependent increase of alpha-1-PI secretion into cell culture medium; this effect was completely reversed after immunoneutralization by the antibodies against IL-2 and IL-8 alpha1-PI secretion was only slightly decreased after treatment with IFN-gamma, while IL-1beta, IL-6 and TNF-alpha had no effect. alpha1-PI secretion correlated well with the expression of this protein in differentiated Caco-2 cells after cytokine treatment, as confirmed by Western blot. Our data imply that, in vitro, alpha1-PI secretion in enterocyte-like Caco-2 cells is up-regulated by IL-2 and IL-8. Our results suggest that both lymphocyte- and macrophage-derived cytokines regulate secretion of the anti-inflammatory protein alpha1-PI in intestinal epithelial cells.

Polyamines arrive in the gut lumen mainly with food. Shortly after a meal, the majority of luminal polyamines disappear from the duodenal and jejunal lumen, by a mechanism of passive diffusion. The majority of luminal polyamines are degraded in the gut before reaching systemic circulation. Hence, there is broad evidence that luminal polyamines are indeed absorbed, distributed throughout the body, and utilized for cellular growth in remote organs and tissues. In addition, luminal polyamines are crucially involved in normal, adaptive and neoplastic growth of the gut per se, and are taken up by normal and neoplastic epithelial cells of the gut mucosa by a tightly regulated and presumably active transport process. Uptake of polyamines into intestinal and colonic epithelial cells is the highest during cell proliferation, and is stimulated by mitogens and peptide growth factors. Understanding the mechanisms of polyamine uptake in neoplastic cells of the gut, as well as the "biodistribution/bioavailability" of luminal polyamines in man, may provide clinically relevant information that can be used in inhibiting cancer cell growth by deprivation of intracellular polyamine pools.

Tributyrin, a prodrug of natural butyrate, has been evaluated with an aim to overcome pharmacokinetic drawbacks of natural butyrate as a drug, i.e., its rapid metabolization and inability to achieve pharmacologic concentrations in neoplastic cells. We studied the effects of tributyrin on growth, differentiation and vitamin D receptor expression in Caco-2 cells, a human colon cancer cell line. Tributyrin was more potent in inhibiting growth and inducing cell differentiation than natural butyrate. The effect was further enhanced after addition of physiologic concentrations of dihydroxycholecalciferol [(OH)2D3]. The synergistic effect of tributyrin and (OH)2D3 in Caco-2 cells was due to tributyrin-induced overexpression of the vitamin D receptor, as measured by reverse transcriptase-polymerase chain reaction. Treatment with tributyrin increased binding of (OH)2D3 to its receptor 1.5-fold, without any change in receptor affinity. We conclude that tributyrin may, at least in part, exert its growth-reducing and differentiation-inducing effect in Caco-2 cells by an upregulation of the vitamin D receptor; this may provide a useful therapeutic approach in chemoprevention and treatment of colorectal cancer by the two nutrients occurring naturally in human diet.

AIM: To study the transepithelial transport characteristics of the polyamine putrescine in human intestinal Caco-2 cell monolayers to elucidate the mechanisms of the putrescine intestinal absorption. METHODS: The transepithelial transport and the cellular accumulation of putrescine was measured using Caco-2 cell monolayers grown on permeable filters. RESULTS: Transepithelial transport of putrescine in physiological concentrations ( > 0.5 mM) from the apical to basolateral side was linear. Intracellular accumulation of putrescine was higher in confluent than in fully differentiated Caco-2 cells, but still negligible (less than 0.5%) of the overall transport across the monolayers in apical to basolateral direction.EGF enhanced putrescine accumulation in Caco-2 cells by four fold, as well as putrescine conversion to spermidine and spermine by enhancing the activity of S adenosylmethionine decarboxylase. However, EGF did not have any significant influence on putrescine flux across the Caco- 2 cell monolayers. Excretion of putrescine from Caco-2 cells into the basolateral medium did not exceed 50 picomoles, while putrescine passive flux from the apical to the basolateral chamber, contributed hundreds of micromoles polyamines to the basolateral chamber. CONCLUSION: Transepithelial transport of putrescine across Caco-2 cell monolayers occurs in passive diffusion, and is not influenced when epithelial cells are stimulated to proliferate by a potent mitogen such as EGF.

Das Mukosaepithel des menschlichen Dunn- und Dickdarms ist durch einen lebenslangen raschen Zellumsatz mit nahezu komplettem Austausch der intestinalen Epithelzellpopulationen innerhalb von 24 bis 96 Stunden gekennzeichnet. Kontinuierliches Wachstum ist somit eine charakteristische Eigenschaft des Dunn- und Dickdarms und eine unerlasliche Voraussetzung, um die intestinale Integritat und Barrierenfunktion zu bewahren. Intestinale Epithelzellen konnen stark vereinfacht in zwei unterschiedliche Kompartimente eingeteilt werden. Ein proliferatives Epithelzellkompartiment ist im Bereich der intestinalen Krypten lokalisiert und raumlich von einem funktionellen Epithelzellkompartiment getrennt, das aus z. T. hochdifferenzierten Epithelzellen im Bereich der intestinalen Villi besteht. Epithelzellen des funktionellen Epithelzellkompartiments migrieren in Richtung Villusspitzen und durchlaufen dabei eine zunehmende Differenzierung und Spezialisierung. Unter physiologischen Bedingungen besteht ein ausgewogenes Gleichgewicht zwischen der Neubildung von intestinalen Epithelzellen durch epitheliale Zellproliferation und dem Verlust von intestinalen Epithelzellen im Rahmen von Zelltod, Apoptose, sowie mechanischer Abschilferung durch verschiedene luminale Faktoren.

Background/Aims: Previous observations have shown that mercapto- and bromo- short-chain fatty acids diminish fatty acid use in colonic epithelium. The aim of this study was to investigate whether this effect is attributable to the inhibition of short-chain fatty acid uptake. Methods: Apical membrane vesicles of rat colonocytes were prepared by a discontinuous sucrose gradient after isolation of membrane caps. [ 14 C]butyrate uptake was measured by rapid filtration technique. Results: Preloading of isolated apical membrane vesicles with bicarbonate or butyrate stimulated [ 14 C]butyrate uptake and resulted in up to fivefold overshoots. Increasing extravesicular butyrate concentrations saturated the bicarbonate-stimulated butyrate uptake with a binding constant of 44.7 ± 5.9 mmol/L and a maximum velocity of 33.2 ± 2.7 nmol · mg protein −1 · 3 s −1 . Intravesicular butyrate uptake was inhibited by addition of 20 mmol/L 3-mercaptopropionate (43.0% ± 5.6%), whereas 2-bromopropionate (13.9% ± 4.1%) and 4-bromobutyrate (22.6% ± 5.3%) did not significantly alter butyrate uptake. Increasing concentrations of 3-mercaptopropionate had a competitive inhibitory effect on butyrate uptake with a binding constant following inhibition of 6.25 ± 0.87 mmol/L and a maximum velocity of 5.82 ± 1.01 nmol · mg protein −1 · 3 s −1 . Conclusions: Butyrate uptake in apical membrane vesicles of rat distal colon is mediated by a low-affinity anion transport system, which can be competitively inhibited by 3-mercaptopropionate but not by 2-bromopropionate and 4-bromobutyrate.

Putrescine transport was investigated in isolated brush border and basolateral membrane vesicles prepared from the rabbit enterocyte. Brush border vesicles were oriented right-side-out and basolateral vesicles inside-out, forming a model representing uptake and extrusion across the intestinal epithelium. Putrescine transport across both membranes was initially rapid, and 66% of the equilibrium uptake was achieved within the first minute. According to osmoplots and measurements at 4 degrees C, 20% of total incorporation presented binding to the membrane. In order to estimate actual uptake into the vesicles, Km was calculated from the differences in putrescine incorporation at 37 degrees C and 4 degrees C, and was 12.7 mumol L-1 for brush border uptake and 38.2 mumol L-1 for basolateral extrusion. Putrescine uptake into brush border and basolateral membrane vesicles was not enhanced in the presence of an Na+ gradient. When Na+ was substituted with an uncharged solute, mannitol, putrescine incorporation was increased, indicating that putrescine uptake is not Na(+)-dependent and that cations might interfere with the carrier. Paraquat and methylglyoxalbis(guanylhydrazone), known to share the polyamine transport system, inhibited putrescine incorporation in both membrane vesicle preparations. Basolateral carrier showed significantly higher sensitivity to cations. We conclude that putrescine uptake across the apical membrane and extrusion across the basolateral membrane of the enterocyte are mediated by two different and independent carriers which differ in their electrical properties.

We report a 60-year-old man with intermittent diarrhea for 15 years which was worsened by cholecystectomy. The results of routine laboratory tests, x-ray studies of the gut, and endoscopy were normal. Fecal fat excretion was normal, but total bile acid excretion was increased in the alkaline feces. The retention of 75SeHCAT was low. Jejunal mucosal biopsy showed normal mucosa. Ileal biopsy was performed at ileoscopy some 10 to 15 cm proximal to the ileocecal valve. Scanning-electron and light microscopy showed a transitional zone between villous atrophy in the distal and normal mucosa in the proximal part of the biopsy specimen. The response of diarrhea to cholestyramine treatment was unequivocal, suggesting bile acid malabsorption. The increased enterohepatic cycling of bile acids after cholecystectomy may be responsible for a decrease in their ileal uptake. Very often, as in our patient, the diarrhea precedes cholecystectomy and is considerably increased by it. Cholecystectomy may unmask a previously unrecognized bile acid transport defect. In our patient, this defect might have been caused by the villous atrophy of the terminal ileum.

Background Deoxycholic acid and other secondary bile acids have long been considered tumour promoters in the colon. However, their effect on cell migration, known to play an important role in colon carcinogenesis, has not been studied so far. Objective To investigate the possible effects of deoxycholic acid on colon cancer-cell migration in culture. Methods Human colon carcinoma cells (Caco-2) were seeded on basement membrane matrix. To evaluate replication-blocked cell migration, we wounded confluent monolayers of cells with a sterile scalpel, and inhibited cell replication with mitomycin C. Immediately after wounding, the cells were exposed to 0-100 micromol/l deoxycholic acid. Migration over 72 h was monitored using a phase contrast microscope. Results Replication-blocked migration was stimulated by deoxycholic acid in a dose-dependent manner, with the maximum effect at 20 micromol/l deoxycholic acid. Enhancement of migration rate was unaffected by immunoneutralization of transforming growth factor beta (a known migration-promoting peptide). However, specific inhibition of protein kinase C markedly inhibited deoxycholic acid-induced Caco-2 cell migration. Conclusion In addition to its well-established role in the enhancement of proliferation, deoxycholic acid also stimulates colon cancer-cell migration along the basement membrane matrix. The mechanism of this stimulation is likely to involve protein kinase C. Deoxycholic acid-stimulated migration might additionally contribute to the tumour-promoting effects of secondary bile acids in the colon.

Three patients are presented with a history of chronic watery diarrhea due to bile acid malabsorption, proved by the tauro-23[75Se]selena-25 homocholic acid test and an unequivocal response to cholestyramine therapy. Fecal fat tests, Schilling tests, and barium studies of the small intestine and colon were all normal. Jejunal biopsies were normal but multiple biopsies of the terminal ileum, performed by retrograde ileoscopy, showed uniform crypt hyperplastic villous atrophy and features of colonic metaplasia with increased mononuclear infiltration of the lamina propria. All 3 patients demonstrated evidence of abnormal immune function, including the presence of serum autoantibodies, circulating immune complexes, and hypocomplementemia. One patient had Sjogren's syndrome. This disorder, which might be immunologically mediated, should be called primary bile acid malabsorption and should be distinguished from other ileal disorders.