Validation of a method for the determination of triphenylmethane dyes in trout and shrimp with superior extraction efficiency.

The described methods are able to analyse the triphenylmethane dyes malachite green (MG), crystal violet (CV) and brilliant green (BG) as well as their leuco metabolites leuco malachite green (LMG), leuco crystal violet (LCV) and leuco brilliant green (LBG) on the basis of a simple and fast extraction. The validation of the methods in two studies without and with a heated ultrasonic treatment during the extraction of fortified trout and shrimp samples was successfully performed applying an in-house validation concept. The evaluation of the relevant validation parameters, e.g. the decision limit CCα, the detection capability CCß, the repeatability, the within-laboratory reproducibility and the recovery for both extraction versions, showed results which fulfil the requirements of Commission Decision 2002/657/EC. The investigation of incurred material of trout containing the above compounds with an additional heated ultrasonic treatment during extraction leads to higher findings of MG and BG. This effect was also confirmed by other laboratories in the framework of a proficiency test. For CV and all three leuco metabolites no increase in the detected amounts could be observed after a heated ultrasonic treatment of the incurred trout material.

In-house validation of a liquid chromatography-tandem mass spectrometry method for the determination of selective androgen receptor modulators (SARMS) in bovine urine.

A sensitive and robust LC-MS/MS method allowing the rapid screening and confirmation of selective androgen receptor modulators in bovine urine was developed and successfully validated according to Commission Decision 2002/657/EC, chapter 3.1.3 'alternative validation', by applying a matrix-comprehensive in-house validation concept. The confirmation of the analytes in the validation samples was achieved both on the basis of the MRM ion ratios as laid down in Commission Decision 2002/657/EC and by comparison of their enhanced product ion (EPI) spectra with a reference mass spectral library by making use of the QTRAP technology. Here, in addition to the MRM survey scan, EPI spectra were generated in a data-dependent way according to an information-dependent acquisition criterion. Moreover, stability studies of the analytes in solution and in matrix according to an isochronous approach proved the stability of the analytes in solution and in matrix for at least the duration of the validation study. To identify factors that have a significant influence on the test method in routine analysis, a factorial effect analysis was performed. To this end, factors considered to be relevant for the method in routine analysis (e.g. operator, storage duration of the extracts before measurement, different cartridge lots and different hydrolysis conditions) were systematically varied on two levels. The examination of the extent to which these factors influence the measurement results of the individual analytes showed that none of the validation factors exerts a significant influence on the measurement results.

Tylosin susceptibility of Staphylococci from bovine mastitis.

Although the 16-membered macrolide tylosin is commonly used for the treatment of bovine mastitis, little information is currently available about the susceptibility of mastitis pathogens to tylosin. In the present study, 112 Staphylococcus aureus and 110 coagulase-negative Staphylococcus (CoNS) spp. isolates from cases of bovine mastitis were tested by broth microdilution and agar disk diffusion with 30 µg tylosin disks. Susceptibility to erythromycin was tested by broth microdilution and disk diffusion using 15 µg disks. Both test populations showed bimodal distributions of minimal inhibitory concentrations (MICs) and zone diameters with eleven S. aureus and eight CoNS isolates showing tylosin MICs of ≥ 256 µg/ml and no zones of growth inhibition around the tylosin 30 µg disks. All 19 isolates with tylosin MICs of ≥ 256 µg/ml were also resistant to erythromycin. For six additional erythromycin-resistant isolates, tylosin MICs of 1-8 µg/ml were observed. One S. aureus and two CoNS isolates showed inducible macrolide resistance. PCR analysis of the 25 erythromycin-resistant staphylococcal isolates identified the resistance genes erm(A), erm(B), erm(C), erm(T), mph(C) and msr(A) alone or in different combinations. An excellent correlation between the results of the different tylosin susceptibility tests (broth microdilution versus disk diffusion) was seen for S. aureus and CoNS isolates. Since tylosin does not induce the expression of the aforementioned erm genes, isolates with an inducible resistance phenotype may - if only tylosin is tested - be falsely classified as tylosin-susceptible. Thus, erythromycin should be tested in parallel and tylosin should only be used for the treatment of infections caused by erythromycin-susceptible staphylococci.

Pheno- and genotypic analysis of antimicrobial resistance properties of Yersinia ruckeri from fish.

Enteric red-mouth disease, caused by Yersinia ruckeri, is an important disease in rainbow trout aquaculture. Antimicrobial agents are frequently used in aquaculture, thereby causing a selective pressure on bacteria from aquatic organisms under which they may develop resistance to antimicrobial agents. In this study, the distribution of minimal inhibitory concentrations (MICs) of antimicrobial agents for 83 clinical and non-clinical epidemiologically unrelated Y. ruckeri isolates from north west Germany was determined. Antimicrobial susceptibility was conducted by broth microdilution at 22 ± 2°C for 24, 28 and 48 h. Incubation for 24h at 22 ± 2°C appeared to be suitable for susceptibility testing of Y. ruckeri. In contrast to other antimicrobial agents tested, enrofloxacin and nalidixic acid showed a bimodal distribution of MICs, with one subpopulation showing lower MICs for enrofloxacin (0.008-0.015 µg/mL) and nalidixic acid (0.25-0.5 µg/mL) and another subpopulation exhibiting elevated MICs of 0.06-0.25 and 8-64 µg/mL, respectively. Isolates showing elevated MICs revealed single amino acid substitutions in the quinolone resistance-determining region (QRDR) of the GyrA protein at positions 83 (Ser83-Arg or -Ile) or 87 (Asn87-Tyr), which raised the MIC values 8- to 32-fold for enrofloxacin or 32- to 128-fold for nalidixic acid. An isolate showing elevated MICs for sulfonamides and trimethoprim harbored a ∼ 8.9 kb plasmid, which carried the genes sul2, strB and a dfrA14 gene cassette integrated into the strA gene. These observations showed that Y. ruckeri isolates were able to develop mutations that reduce their susceptibility to (fluoro)quinolones and to acquire plasmid-borne resistance genes.

Analysis of extended-spectrum-ß-lactamase-producing Escherichia coli isolates collected in the GERM-Vet monitoring programme.

OBJECTIVES: The aims of this study were (i) to detect extended-spectrum ß-lactamase (ESBL) genes among 1378 Escherichia coli isolates from defined disease conditions of companion and farm animals and (ii) to determine the localization and organization of ESBL genes. METHODS: E. coli isolates from the German resistance monitoring programme GERM-Vet were included in the study. Plasmids were transferred by conjugation or transformation and typed by PCR-based replicon typing. ESBL genes were detected by PCR; the complete ESBL genes and their flanking regions were sequenced by primer walking. Phylogenetic grouping and multilocus sequence typing (MLST) were performed for all ESBL-producing E. coli isolates. RESULTS: Of the 27 ESBL-producing E. coli isolates detected, 22 carried blaCTX-M-1 genes on IncN (n = 16), IncF (n = 3), IncI1 (n = 2) or multireplicon (n = 1) plasmids. A blaCTX-M-3 gene was located on an IncN plasmid and a blaCTX-M-15 gene was located on an IncF plasmid. A multireplicon plasmid and an IncHI1 plasmid harboured blaCTX-M-2. A blaTEM-52c gene was identified within Tn2 on an IncI1 plasmid. The blaCTX-M genes located within the same or related genetic contexts showed differences due to the integration of insertion sequences. Various MLST types were detected, with ST10 (n = 7), ST167 (n = 4) and ST100 (n = 3) being the most common. CONCLUSIONS: This study showed that the blaCTX-M-1 gene is the predominant ESBL gene among E. coli isolates from diseased animals in Germany and a considerable structural heterogeneity was found in the regions flanking the blaCTX-M-1 gene. Insertion sequences, transposons and recombination events are likely to be involved in alterations of the ESBL gene regions.

A proposal of interpretive criteria for cefoperazone applicable to bovine mastitis pathogens.

The correct assessment of mastitis pathogens for their susceptibility/resistance to cefoperazone is currently hampered by the lack of harmonized test conditions and interpretive criteria. The aim of this study was to provide a proposal for clinical breakpoints of cefoperazone which are applicable to Staphylococcus aureus, coagulase-negative staphylococci, Escherichia coli, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis from cases of bovine mastitis and better reflect the situation in the bovine udder than breakpoints adopted from human medicine. For this, pharmacological data and clinical efficacy data of the documents submitted for approval of cefoperazone have been revisited. In addition, 1086 bacterial pathogens of the aforementioned six species/groups collected in Germany and in the USA during recent years were tested in parallel for their cefoperazone MICs and the zone diameters using a 75 µg disk. Subsequently, MICs were plotted against zone diameters. Based on the pharmacological data, the clinical efficacy and the microbiological data, a proposal was made for veterinary-specific breakpoints which classify members of the aforementioned species/groups as (a) susceptible to cefoperazone when their MIC is ≤ 2 µg/ml and their zone diameters are ≥ 27 mm (staphylococci or E. coli) or ≥ 21 mm (streptococci), (b) intermediate when their MIC is 4 µg/ml and their zone diameters are 22-26 mm (staphylococci or E. coli) or 16-20mm (streptococci), and (c) resistant when their MIC is ≥ 8 µg/ml and their zone diameters are ≤ 21 mm (staphylococci or E. coli) or ≤ 15 mm (streptococci).

Transforming growth factor-ß, a whey protein component, strengthens the intestinal barrier by upregulating claudin-4 in HT-29/B6 cells.

TGFß (isoforms 1-3) has barrier-protective effects in the intestine. The mechanisms involved in regulating tight junction protein expression are poorly understood. The aim of this study was to elucidate TGFß-dependent protective effects with special attention to promoter regulation of tight junction proteins using the HT-29/B6 cell model. In addition, the effects of whey protein concentrate 1 (WPC1), a natural source of TGFß in human nutrition, were examined. For this purpose, the claudin-4 promoter was cloned and tested for its activity. It exhibited transactivation in response to TGFß1, which was intensified when Smad-4 was cotransfected, indicating a Smad-4-dependent regulatory component. Shortening and mutation of the promoter altered and attenuated this effect. WPC1 induced an increase in the claudin-4 protein level and resistance of HT-29/B6 cell monolayers. Anti-TGFß(1-3) antibodies blocked these whey protein effects, suggesting that a main part of this function was mediated through TGFß. This effect was observed on intact monolayers as well as when barrier function was impaired by preexposure to IFNγ. In conclusion, TGFß1 affects claudin-4 gene expression via Smad-4-dependent and -independent transcriptional regulation, resulting in barrier protection, a cytokine effect that is also found in whey protein concentrates used in enteral nutrition.

Norovirus non-structural protein p20 leads to impaired restitution of epithelial defects by inhibition of actin cytoskeleton remodelling.

OBJECTIVE: Norovirus is the most common cause of acute gastroenteritis in humans worldwide. Typical symptoms are vomiting, nausea and severe watery diarrhea. Because of the lack of cell lines susceptible to human norovirus infection, pathomechanisms and replication cycle are largely unknown. Here, we address the issue of how norovirus infection could lead to epithelial barrier dysfunction. MATERIAL AND METHODS: Expression of the non-structural norovirus protein p20 in the epithelial cell line HT-29/B6 was activated through a tetracycline sensitive promoter. Tight junction proteins were studied by Western blot and confocal laser scanning microscopy. Apoptoses were detected in TUNEL stainings. Epithelial restitution was monitored by conductance scanning after induction of single cell lesions. RESULTS: Changes in the expression or localization of the tight junction proteins occludin and/or claudin-1, -2,- 3, -4, -5, -7 and -8 could be ruled out to mediate epithelial barrier modulation. Cell motility was also unaltered by p20. Investigation of epithelial apoptosis revealed an accumulation of apoptic cells in epithelial monolayers after induction of p20 expression. In epithelial cell restitution assays, an arrest was identified in p20 expressing cells. Fluorescence microscopy revealed an inability for condensation and redistribution of cellular actin, which led to a reduced transepithelial electrical resistance. CONCLUSIONS: Functional data for norovirus protein p20 suggest a role in modulation of the actin cytoskeleton leading to barrier dysfunction through impairment of restitution of epithelial defects.

A practical approach to screen for authorised and unauthorised genetically modified plants.

In routine analysis, screening methods based on real-time PCR are most commonly used for the detection of genetically modified (GM) plant material in food and feed. In this paper, it is shown that the combination of five DNA target sequences can be used as a universal screening approach for at least 81 GM plant events authorised or unauthorised for placing on the market and described in publicly available databases. Except for maize event LY038, soybean events DP-305423 and BPS-CV127-9 and cotton event 281-24-236 x 3006-210-23, at least one of the five genetic elements has been inserted in these GM plants and is targeted by this screening approach. For the detection of these sequences, fully validated real-time PCR methods have been selected. A screening table is presented that describes the presence or absence of the target sequences for most of the listed GM plants. These data have been verified either theoretically according to available databases or experimentally using available reference materials. The screening table will be updated regularly by a network of German enforcement laboratories.

Diversity of antimicrobial resistance pheno- and genotypes of methicillin-resistant Staphylococcus aureus ST398 from diseased swine.

OBJECTIVES: Fifty-four methicillin-resistant Staphylococcus aureus (MRSA) ST398 isolates from unrelated diseased swine collected all over Germany were comparatively investigated for their antimicrobial resistance and virulence properties, and for their genomic relatedness. METHODS: MICs of 30 antimicrobial agents were determined by broth microdilution. Resistance and virulence genes were detected via a diagnostic DNA microarray and specific PCRs. The genomic relationships were determined by ApaI-PFGE, spa typing and SCCmec typing. RESULTS: Twenty-two distinct resistance patterns were observed. All 54 isolates were tetracycline resistant, mediated by tet(M), tet(K) and/or tet(L), with 14 isolates being only resistant to beta-lactam antibiotics and tetracyclines. Trimethoprim resistance, seen in 28 isolates, was mostly due to the gene dfrK or dfrG. Among the 24 macrolide/lincosamide-resistant isolates, the genes erm(A), erm(B) and/or erm(C) were detected. The two chloramphenicol/florfenicol-resistant isolates harboured the gene fexA. The eight gentamicin-resistant isolates carried the gene aacA/aphD. Fifty-three isolates harboured SCCmec type V elements while the remaining one carried mecA and ugpQ, but no recombinase genes. All isolates were PVL negative, but one and three isolates, respectively, were positive for the enterotoxin B and enterotoxin K and Q genes. Eight different spa types were identified with t011 being the most predominant. Six ApaI-PFGE clusters with up to nine individual patterns were detected. CONCLUSIONS: MRSA ST398 isolates varied slightly in their virulence properties and spa types but differed distinctly in their antimicrobial resistance pheno- and genotypes as well as their ApaI-PFGE patterns. These data underline the ability of ST398 to acquire genetic material that might increase antimicrobial resistance and virulence.

Quercetin enhances epithelial barrier function and increases claudin-4 expression in Caco-2 cells.

Quercetin is the most abundant flavonoid and is assumed to have positive effects on the gastrointestinal mucosa after dietary intake. The aim of the study was to analyze the influence of quercetin on intestinal barrier function using the human colonic epithelial cell line Caco-2. Transepithelial resistance (R(t)), tracer fluxes of [(3)H]-mannitol, 22Na+, and 36Cl- as well as electrogenic ion transport were determined in Ussing chambers. Expression of tight junction (TJ) proteins and mRNA was analyzed in Western blots and quantitative RT-PCR, respectively. Regulation of transcription was analyzed by reporter gene assay. Cellular distribution of TJ proteins was examined by confocal laser scanning microscopy (LSM). Apoptotic rate was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. Quercetin induced a dose-dependent increase of R(t) persisting for >2 d. Daily addition of quercetin was able to perpetuate the effect, which was seen whether quercetin was added apically or to the basolateral compartment. Parallel to the R(t) increase, quercetin induced a strong increase of the TJ protein claudin-4 but not of other claudins. Confocal LSM showed a localization of claudin-4 in TJ. Apoptotic rate was not affected by quercetin. Consistent with these changes, fluxes of Na+ and Cl-, but not of mannitol, were reduced. Reporter gene assays revealed a stimulatory effect of quercetin on claudin-4 transcription. The flavonoid quercetin enhances barrier function via transcriptional expression regulation of the TJ protein claudin-4, which represents an important protective effect of this food component against barrier disturbance in intestinal inflammation.

Altered permeability in inflammatory bowel disease: pathophysiology and clinical implications.

PURPOSE OF REVIEW: To present the mechanisms behind barrier disturbance in inflammatory bowel disease and their functional consequences. RECENT FINDINGS: A reduction in tight junction strands, strand breaks and alteration of tight junction protein content and composition characterize Crohn's disease. In ulcerative colitis, epithelial leaks appear early as a result of microerosions, upregulated epithelial apoptosis and tight junction protein changes with pronounced increases in claudin-2. T-helper type 1 cytokine effects by interferon-gamma and tumour necrosis factor alpha are important for epithelial damage in Crohn's disease. Interleukin-13 is the key effector cytokine in ulcerative colitis, stimulating epithelial cell apoptosis, and can upregulate claudin-2 expression. Together with interleukin-13-induced epithelial restitution arrest, this may explain why ulcer lesions occur in early stages of ulcerative colitis but are only observed in advanced inflammatory stages in Crohn's disease. SUMMARY: Barrier dysfunction in inflammatory bowel disease contributes to diarrhea by a leak flux mechanism and can cause mucosal inflammation secondary to luminal antigen uptake. Barrier abnormalities, such as epithelial tight junction changes and apoptotic leaks, gross mucosal lesions, and epithelial restitution arrest are responsible for these abnormalities and are the result of immune dysregulation. Studying the underlying mechanisms is important in understanding the pathophysiology of inflammatory bowel disease and developing therapeutic strategies.

Butyrate induces intestinal sodium absorption via Sp3-mediated transcriptional up-regulation of epithelial sodium channels.

BACKGROUND & AIMS: The epithelial sodium channel (ENaC) is the rate-limiting factor for colonic electrogenic sodium absorption. This study aimed to investigate ENaC regulation by butyrate, a short-chain fatty acid (SCFA) produced by intestinal bacteria. METHODS: ENaC was examined in HT-29/B6 cells and glucocorticoid receptor(GR)-transfected HT-29/B6 cells (HT-29/B6-GR) by reverse-transcription polymerase chain reaction, real-time polymerase chain reaction, and confocal microscopy. ENaC promoters were investigated by deletion/mutation analysis, electrophoretic mobility shift assays, and quantitative chromatin immunoprecipitation. Sodium transport of HT-29/B6-GR cells and rat distal colon was quantified in Ussing chambers. RESULTS: Butyrate up-regulated beta- and gamma-ENaC mRNA expression in HT-29/B6 cells and induced transcription from beta- and gamma-ENaC promoter constructs. The gamma-ENaC promoter could also be induced by the SCFA propionate but not by acetate. Deletion/mutation assays revealed that activation of the gamma-ENaC promoter depended on 2 GC boxes, which were shown to bind Sp1 and Sp3 in vitro. Although both transcription factors increased butyrate-mediated gamma-ENaC transcription upon overexpression, chromatin immunoprecipitation revealed that only Sp3 binds to the gamma-ENaC promoter in vivo and that Sp3 binding is enhanced by butyrate. Transcriptional ENaC induction by butyrate led to synthesis of gamma-ENaC subunits, but correct targeting of ENaC channels to the apical cell membrane was dependent on corticosteroid hormones. Finally, butyrate substantially increased electrogenic sodium absorption via ENaC in the presence of corticosteroid hormones in HT-29/B6-GR cells and in rat distal colon. CONCLUSIONS: Concerted action of SCFA and corticosteroid hormones is required for induction of ENaC and maintenance of intestinal electrogenic sodium absorption.

Restoration of ENaC expression by glucocorticoid receptor transfection in human HT-29/B6 colon cells.

The epithelial sodium channel (ENaC) controls colonic sodium absorption. So far, investigation of ENaC was limited by an unexplained lack of steroid-dependent ENaC expression in cultured intestinal cells, which we aimed to resolve. HT-29/B6 cells constitutively expressed the alpha-ENaC subunit, while beta- and gamma-ENaC subunits could not be detected due to deficient basal as well as corticosteroid-induced transcription. This was due to a lack of expression of both activating and inhibiting isoforms of glucocorticoid receptor (GR-alpha, -beta) and mineralocorticoid receptor. Stable transfection of GR-alpha restored intestine-specific glucocorticoid upregulation of beta- and gamma-ENaC in HT-29/B6 cells, which was followed by intact targeting of ENaC channels to the apical cell membrane and dose-dependent induction of electrogenic sodium absorption. In conclusion, ENaC deficiency is due to a lack of steroid receptors and not the consequence of a crypt-like phenotype of cultured intestinal cells. By stable GR transfection we obtained a model, in which ENaC regulation can be studied.

Contribution of claudin-5 to barrier properties in tight junctions of epithelial cells.

Claudin-5 is a transmembrane protein reported to be primarily present in tight junctions of endothelia. Unexpectedly, we found expression of claudin-5 in HT-29/B6 cells, an epithelial cell line derived from human colon. Confocal microscopy showed colocalization of claudin-5 with occludin, indicating its presence in the tight junctions. By contrast, claudin-5 was absent in the human colonic cell line Caco-2 and in Madin-Darby canine kidney cells (MDCK sub-clones C7 and C11), an epithelial cell line derived from the collecting duct. To determine the contribution of claudin-5 to tight junctional permeability in cells of human origin, stable transfection of Caco-2 with FLAG-claudin-5 cDNA was performed. In addition, clone MDCK-C7 was transfected. Synthesis of the exogenous FLAG-claudin-5 was verified by Western blot analysis and confocal fluorescent imaging by employing FLAG-specific antibody. FLAG-claudin-5 was detected in transfected cells in colocalization with occludin, whereas cells transfected with the vector alone did not exhibit specific signals. Resistance measurements and mannitol fluxes after stable transfection with claudin-5 cDNA revealed a marked increase of barrier function in cells of low genuine transepithelial resistance (Caco-2). By contrast, no changes of barrier properties were detected in cells with a high transepithelial resistance (MDCK-C7) after stable transfection with claudin-5 cDNA. We conclude that claudin-5 is present in epithelial cells of colonic origin and that it contributes to some extent to the paracellular seal. Claudin-5 may thus be classified as a tight-junctional protein capable of contributing to the "sealing" of the tight junction.

Interleukin-13 is the key effector Th2 cytokine in ulcerative colitis that affects epithelial tight junctions, apoptosis, and cell restitution.

BACKGROUND & AIMS: Ulcerative colitis (UC) is characterized by a Th2 immune response with inflammation and epithelial barrier dysfunction. So far, Th2 cytokines have not been shown to directly influence epithelial barrier function. METHODS: Lamina propria mononuclear cells (LPMCs) were stimulated and interleukin (IL)-13 was measured by enzyme-linked immunosorbent assay. Functional IL-13 and IL-4 effects were studied on HT-29/B6 colonic epithelial cells in Ussing chambers and by conductance scanning. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays. IL-13/IL-4 receptors were analyzed by reverse-transcription polymerase chain reaction and immunofluorescence. Western blotting combined with immunofluorescence was used to detect tight junction proteins. Furthermore, restitution velocity was measured. Finally, mucosal biopsy specimens from patients with UC were compared with cultured cells for these features. RESULTS: LPMCs from patients with UC produced large amounts of IL-13 (985 +/- 73 pg/mL), much more than from controls or patients with Crohn's disease. IL-13Ralpha1 and IL-4Ralpha receptors were present in HT-29/B6 cells and colonic epithelial cells of control patients and patients with UC. IL-13 had a dose-dependent effect on transepithelial resistance of HT-29/B6 monolayers (reduction to 60% +/- 4%), whereas IL-4 had no effect. This was due to an increased number of apoptotic cells (5.6-fold +/- 0.9-fold) and an increased expression of the pore-forming tight junction protein claudin-2 to 295% +/- 37%, both of which contributed equally. Finally, epithelial restitution velocity decreased from 15.1 +/- 0.6 to 10.6 +/- 0.5 microm/h after treatment with IL-13. Parallel changes were observed in human samples, with an increase in claudin-2 expression to 956% +/- 252%. CONCLUSIONS: IL-13 was identified as an important effector cytokine in UC that impairs epithelial barrier function by affecting epithelial apoptosis, tight junctions, and restitution velocity.

Occludin as direct target for glucocorticoid-induced improvement of blood-brain barrier properties in a murine in vitro system.

Homeostasis of the central nervous system (CNS) microenvironment is essential for its normal function. It is maintained by the blood-brain barrier (BBB) which regulates the transport of molecules from blood into brain and backwards. The integrity of the BBB is compromised in many disorders of the human CNS; therapeutical strategies for several of these diseases include treatment with glucocorticoids, but the molecular basis of how glucocorticoids regulate BBB permeability is not understood. Here, we report the generation and characterization of a murine immortalized brain (cerebral) capillary endothelial (cEND) cell line which expresses the BBB marker occludin at intercellular tight junctions (TJ). Hydrocortisone at physiological concentrations induced upregulation of occludin, accompanied by a threefold enhancement of transendothelial electrical resistance to values up to 1000 Omegacm2. Insulin enhanced the glucocorticoid response. At the molecular level, hydrocortisone induces increase of occludin at protein and mRNA levels by activation of the glucocorticoid receptor (GR) and its binding to putative glucocorticoid responsive elements in the occludin promoter. At the same time, insulin potentiated the ligand-dependent GR transactivation via induction of the GR in this in vitro system. This study thus provides insights into the molecular processes of barrier genesis, and may help to elucidate mechanisms of brain pathology at the microvascular level.

Cytokine-dependent transcriptional down-regulation of epithelial sodium channel in ulcerative colitis.

BACKGROUND & AIMS: The main limiting factor for sodium absorption in distal colon is the amiloride-sensitive epithelial sodium channel (ENaC). This study aimed to characterize mechanisms involved in the dysregulation of ENaC expression in ulcerative colitis (UC). METHODS: Epithelial preparations from surgically removed inflamed and control sigmoid colons were used. Active electrogenic Na(+) transport (J(Na)) was determined after 8-hour aldosterone stimulation in Ussing-chambers (corrected for the altered epithelial/subepithelial resistance ratio). Subsequently, ENaC alpha-, beta-, and gamma-subunits were analyzed immunohistochemically and in Western and Northern blots (corrected for the inflammatory increase in subepithelial protein content). To study gene regulation, the promoters of beta- and gamma-ENaC were analyzed in reporter gene assays. RESULTS: In controls, aldosterone stimulated J(Na) and induced ENaC beta- and gamma-subunit expression, whereas this response was virtually abolished in UC. Preservation of surface epithelium in UC was indicated by unchanged ENaC alpha-subunit expression, which points also against a mere immaturity or epithelial cell loss. Inhibition of electrogenic sodium transport as well as beta- and gamma-ENaC mRNA expression could be mimicked in control colon by in vitro preexposure for 8 hours to tumor necrosis factor alpha and interferon gamma. Promoter analysis revealed that down-regulation of beta- and gamma-ENaC gene expression was primarily induced by tumor necrosis factor alpha. CONCLUSIONS: We conclude that, in UC, elevated proinflammatory cytokines selectively impair beta- and gamma-ENaC expression, which contributes to diarrhea by reducing colonic sodium absorption.

IL-1beta and TNFalpha regulate sodium absorption in rat distal colon.

The epithelial Na+ channel (ENaC) provides the main absorptive pathway of the distal large intestine. This study aimed to characterize regulatory influences of cytokines in rat late distal colon. After 6 h incubation with either IL1beta, TNFalpha, IFNgamma, or combinations of TNFalpha and IFNgamma, ENaC was measured as electrogenic Na+ transport after 8 h induction by 3 nM aldosterone (JNa) in totally stripped specimens in the Ussing chamber. Subsequently, alpha-, beta-, and gamma-ENaC subunit mRNAs were analyzed by Northern blotting. The gamma-ENaC promoter was cloned and characterized by reporter gene assays. IL-1beta and TNFalpha, but not interferon-gamma, decreased JNa. In parallel, beta- and gamma-ENaC transcription was inhibited, whereas alpha-ENaC was unaffected. gamma-ENaC promoter activity was inhibited by IL-1beta and TNFalpha but not by IFNgamma. We conclude that the pro-inflammatory cytokines IL-1beta and TNFalpha inhibit electrogenic sodium absorption in rat distal colon by mRNA expression regulation of the beta- and gamma-ENaC subunits.