Comparison of a new microbiological assay with a standard high-performance liquid chromatographic method for determination of vitamin B6 in serum.

BACKGROUND: A high-performance liquid chromatographic (HPLC) assay for vitamin B6 in human serum was compared with a novel microbiological assay (ID-Vit) that uses microtitre plates precoated with a specific microorganism, thus avoiding numerous problems associated with the use of stock cultures utilized by common other microbial assay mit B6. METHODS: Data obtained using HPLC were compared with 1D-Vit results in 170 healthy individuals and in 68 patients with coronary artery disease (CAD, 37 with acute coronary syndrome [ACS], 31 with stable CAD). Regression and Bland-Altman analysis were performed. Homocysteine in CAD patients was measured by HPLC. RESULTS: The ID-Vit assay correlated well with the HPLC assay (Pearson's r = 0.89 [p < 0.0001] in healthy and 0.82 [p < 0.001] in CAD individuals). Bland-Altman analyses revealed good agreement between the results of both methods in both cohorts, with > or = 95% of all values grouping within the lines of agreement. In CAD patients, mean homocysteine values did not differ between stable CAD and ACS and were normal. Thirty-seven percent of CAD patients had estimated glomerular filtration rates (GFR) below 60 mL/min/1.73m2. GFR correlated inversely with homocysteine levels (r = -0.80, p < 0.001) whereas neither HPLC nor ID-Vit values for B6 did. CONCLUSIONS: ID-Vit assay and the HPLC standard are in very good agreement. The new assay can easily be automated and is less laborious than common microbiological assays. The lack of correlation between B6 vitamin and homocysteine can be accounted for by the fact that mean vitamin B6 in our CAD patients was in the normal range and that a relevant percentage of patients had chronic renal disease.

The role of adipokines in the improvement of diabetic and cardiovascular risk factors within a 52-week weight-loss programme for obesity.

BACKGROUND: Obesity is an independent risk factor for cardiovascular disease and diabetes weight reduction not only reduces the risk for these diseases but leads to an alteration of the circulating adipokine levels. The aim of our study was to evaluate the effect of weight loss and lifestyle changes implemented in the form of the interdisciplinary weight management programme Optifast52® on cardiovascular and diabetic risk factors and on key adipokines. METHODS: 72 morbidly obese patients were included in the programme, which consisted of a very low-calorie diet followed by incremental food introduction and dietary stabilisation, accompanied by medical surveillance, physical activity, dietary counselling and psychological support. At baseline, and after 14, 26 and 49 weeks, risk factor profiles and adipokine levels were evaluated. RESULTS: 43 patients completed the programme with an average weight reduction of about 20%. Significant improvement was observed in the lipid and diabetic laboratory panels of all patients. In addition, adiponectin levels increased significantly (7.79 vs. 12.38µg/ml, p<0.001), while leptin levels decreased (7.29 vs 3.09ng/ml, p<0.001) during the course of the programme. CONCLUSION: In this study, Optifast52®, a multidisciplinary programme focusing on diet and lifestyle changes, was found not only to affect a decrease in parameters associated with diabetes and cardiovascular disease, but also to ameliorate in part the obesity-related imbalance of pro- and anti-inflammatory adipokines.

PPARgamma is involved in mesalazine-mediated induction of apoptosis and inhibition of cell growth in colon cancer cells.

PURPOSE: Mesalazine has been identified as a candidate chemopreventive agent in colon cancer prophylaxis because of its pro-apoptotic and anti-proliferative effects. However, the precise mechanisms of action are not entirely understood. The aim of our study was to investigate the involvement of peroxisome proliferator-activated receptor gamma (PPARgamma) in mesalazine's anticarcinogenic actions in colorectal cancer cells. EXPERIMENTAL DESIGN: The effects of mesalazine on cell cycle distribution, cell count, proliferation and caspase-mediated apoptosis were examined in Caco-2, HT-29 and HCT-116 cells used as wild-type, dominant-negative PPARgamma mutant and empty vector cultures. We focused on caspase-3 activity, cleavage of poly(ADP-ribose) polymerase (PARP), caspase-8 and caspase-9, as well as on expression of survivin, X-linked inhibitor of apoptosis (Xiap), phosphatase and tensin homolog deleted from chromosome ten (PTEN) and c-Myc. Techniques employed included transfection assays, immunoblotting, flow cytometry analysis, colorimetric and fluorometric assays. RESULTS: Mesalazine caused a time- and dose-dependent decrease in both cell growth and proliferation. Growth inhibition was accompanied by a G1/G0 arrest, a significant increase in PTEN, caspase-3 activity, cleavage of PARP and caspase-8, whereas the expressions of Xiap, survivin and c-Myc were decreased simultaneously. Cleavage of caspase-9 was not observed. Moreover, PPARgamma expression and activity were elevated. The growth-inhibitory effect of mesalazine was partially reduced in dominant-negative PPARgamma mutant cells, whereas the expression of c-Myc was not affected. Mesalazine-mediated increased caspase-3 activity, the expression of PTEN, cleavage of PARP and caspase-8 as well as reduced levels of survivin and Xiap were completely abolished in the PPARgamma mutant cell lines. CONCLUSION: This study clearly demonstrates that mesalazine-mediated pro-apoptotic and anti-proliferative actions are regulated via PPARgamma-dependent and -independent pathways in colonocytes.

Influence of dTMP on the phenotypic appearance and intracellular persistence of Staphylococcus aureus.

Thymidine-dependent small-colony variants (SCVs) of Staphylococcus aureus are frequently associated with persistent and recurrent infections in cystic fibrosis patients. The phenotypic appearance of S. aureus SCVs or normal-colony variants (NCVs) is postulated to be affected by the intracellular amount of dTMP. This hypothesis was proven by metabolic pathway assays revealing altered intracellular dTMP concentrations, followed by investigation of the associated phenotype. Inhibition of the staphylococcal thymidylate synthase, which generated intracellular dTMP from dUMP, using 5-fluorouracil and co-trimoxazole resulted in an SCV phenotype. Inhibition of a nucleoside transporter, which provided the bacterial cell with extracellular thymidine, caused growth inhibition of SCVs. In turn, reversion of SCVs to NCVs was achieved by supplying extracellular dTMP. High-performance liquid chromatography additionally confirmed the intracellular lack of dTMP in SCVs, in contrast to NCVs. Moreover, the dTMP concentration is postulated to influence the intracellular persistence of S. aureus. Cell culture experiments with cystic fibrosis cells revealed that clinical and co-trimoxazole-induced SCVs with a diminished amount of dTMP showed significantly better intracellular persistence than NCVs. In conclusion, these results show that the dTMP concentration plays a key role in both the phenotypic appearance and the intracellular persistence of S. aureus.

The dietary histone deacetylase inhibitor sulforaphane induces human beta-defensin-2 in intestinal epithelial cells.

Antimicrobial peptides like human beta-defensin-2 (HBD-2) play an important role in the innate immune system protecting the intestinal mucosa against bacterial invasion. The dietary histone deacetylase (HDAC) inhibitors sulforaphane (SFN) and butyrate have received a great deal of attention because of their ability to simultaneously modulate multiple cellular targets involved in cellular protection. In this study the influence of SFN and butyrate on HBD-2 expression as well as the molecular pathways involved in SFN-mediated induction of HBD-2 were scrutinized. Treatment of Caco-2, HT-29 and SW480 cells with SFN led to a time- and dose-dependent upregulation of HBD-2 mRNA expression as determined by semi-quantitative reverse transcription-polymerase chain reaction. Moreover, HBD-2 protein production increased in response to SFN, measured by enzyme-linked immunosorbent assay. Induction of HBD-2 was also observed in response to butyrate. Immunofluorescence analysis revealed that the protein was localized in the cytosol. Coincubation of SFN with a vitamin D receptor (VDR), or an extracellular-regulated kinase 1/2 or a nuclear factor-kappaB inhibitor all reduced HBD-2 mRNA upregulation. In contrast, transfection of cells with a dominant-negative peroxisome proliferator-activated receptor gamma (PPARgamma) mutant vector to inhibit PPARgamma wild-type action and inhibition of p38 mitogen-activated protein kinase (MAPK) signalling did not affect SFN-mediated upregulation of HBD-2 mRNA. Moreover, SFN induced the expression of VDR, PPARgamma and phosphorylated ERK1/2 but did not affect p38 MAPK activation. The data clearly demonstrate for the first time that the dietary HDAC inhibitor SFN is able to induce antimicrobial peptides in colonocytes. In this process HBD-2 expression is regulated via VDR, mitogen-activated protein kinase kinase/extracellular-regulated kinase and nuclear factor-kappaB signalling.

Comparison of an established simple office-based immunological FOBT with fecal tumor pyruvate kinase type M2 (M2-PK) for colorectal cancer screening: prospective multicenter study.

OBJECTIVES: The immunological fecal occult blood test (IFOBT) has established itself as a more precise marker for colorectal cancer (CRC) screening than traditional guaiac-based FOBT. The simpler, cheaper, and more convenient newer office-based IFOBTs have been validated for diagnosing CRC. Dimeric isoenzyme of pyruvate kinase, M2-PK, expressed by tumor cells, has as well been proposed as a screening tool for CRC. This is the first study comparing fecal M2-PK as a screening biomarker for CRC against previously evaluated office-based IFOBT and colonoscopy. METHODS: Six hundred forty consecutive subjects (symptomatic, as well as for CRC screening) referred for colonoscopy for various indications across five centers in Germany provided the stool samples for performing M2-PK and an immunochemical FOB strip test. The IFOBT used was a rapid immunochromatographic assay for detection of fecal hemoglobin. For M2-PK, a commercially available sandwich enzyme-linked immunosorbent assay (ELISA) was used. The M2-PK test needs 6 h, while the office-based test can be read in just 10 min and is five times cheaper. RESULTS: Office-based IFOBT had sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and positive and negative likelihood ratios (LR) of 64.5, 96.3, 72.0, 94.9, 17.5, and 0.4 for diagnosing colorectal neoplasia (CRN), while the above performance characteristics for M2-PK at a cutoff value of 4 U/mL were 72.4, 73.8, 29.0, 94.8, 2.8, and 0.8 respectively. CONCLUSIONS: This office-based IFOBT was found to have significantly higher specificity, PPV, and positive LR as compared with M2-PK. IFOBT proved to be a convenient, noncumbersome, quick, and cheap tool in patients with above-average risk for detection of CRN.

Role of nuclear hormone receptors in butyrate-mediated up-regulation of the antimicrobial peptide cathelicidin in epithelial colorectal cells.

BACKGROUND AND AIMS: The human cathelicidin (LL-37) is one of the major antimicrobial peptides of the non-specific innate immune system in the intestinal tract. Altered expression has been associated with gastrointestinal disease. Recent studies demonstrated that butyrate induces LL-37 mRNA in colonic epithelial cells, however the underlying molecular mechanisms have not been elucidated. The objective of this study was to investigate the regulatory pathways involved in butyrate-induced up-regulation of LL-37. METHODS AND RESULTS: Treatment of Caco-2 and HT-29 cells with butyrate led to a time-dependent up-regulation of LL-37 mRNA expression as determined by semi-quantitative RT-PCR. Up-regulation of LL-37 mRNA by butyrate was subsequently followed by an increase in LL-37 protein expression as observed by immunofluorescence. Co-incubation of butyrate with a VDR, p38 MAPK, ERK 1/2 and TGF-beta1 receptor kinase inhibitor all reduced butyrate-mediated LL-37 mRNA up-regulation. In contrast, transfection of Caco-2 cells with a dominant-negative PPARgamma mutant vector did not affect butyrate-mediated up-regulation of LL-37 mRNA. CONCLUSION: Our results clearly demonstrate that butyrate-mediated up-regulation of LL-37 is influenced by several signalling pathways and receptors including MAPKs as well as VDR and TGF-beta1, but not by PPARgamma. These data may provide new opportunities in the treatment of gastrointestinal diseases.

Involvement of different nuclear hormone receptors in butyrate-mediated inhibition of inducible NF kappa B signalling.

BACKGROUND: NF kappa B plays a major role in the control of immune responses and inflammation. Recently, butyrate has not only been demonstrated to suppress NF kappa B activation in colorectal cancer cells, but also to modulate the activity and expression of the Peroxisome-Proliferator-Activated-Receptor gamma (PPAR gamma) and the vitamin D receptor (VDR). Therefore, we investigated a putative involvement of both receptors in butyrate-mediated inhibition of inducible NF kappa B signalling. RESULTS: Treatment of HT-29 cells with butyrate attenuated basal p50 as well as TNFalpha- and LPS-induced p50 and p65 NF kappa B dimer activity in the nucleus as measured by transcription factor assay. Cytosolic expression of I kappa B alpha protein was reduced by butyrate, and TNFalpha but not by LPS. Challenge of cells with the VDR antagonist ZK191732 up-regulated basal NF kappa B activity by decreasing I kappa B alpha simultaneously, while basal signalling was not influenced by the PPAR gamma inhibitor GW9662. Pre-treatment with ZK191732 reduced the inhibitory effect of butyrate on NF kappa B activation caused by TNFalpha whereas no activation was noted in transfected dominant-negative PPAR gamma mutant vector cells. Adversely, the inhibitory effect of butyrate on NF kappa B activity induced by LPS was almost reversed in dominant-negative PPAR gamma mutant cells while pre-incubation of ZK191732 did not affect butyrate-mediated attenuation of LPS-induced NF kappa B signalling. CONCLUSION: These findings provide evidence for the involvement of the nuclear hormone receptors PPAR gamma and VDR in butyrate-mediated inhibition of inducible NF kappa B activation dependent on the stimulated signalling pathway. Moreover, VDR appears to play an inhibitory role in the regulation of basal NF kappa B signalling.

Peroxisome proliferator-activated receptor gamma as a molecular target of resveratrol-induced modulation of polyamine metabolism.

Previous results indicate that the polyphenol resveratrol inhibits cell growth of colon carcinoma cells via modulation of polyamine metabolic key enzymes. The aim of this work was to specify the underlying molecular mechanisms and to identify a possible role of transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma). Cell growth was determined by bromodeoxyuridine incorporation and crystal violet staining. Protein levels were examined by Western blot analysis. Spermine/spermidine acetyltransferase (SSAT) activity was determined by a radiochemical assay. PPARgamma ligand-dependent transcriptional activity was measured by a luciferase assay. A dominant-negative PPARgamma mutant was transfected in Caco-2 cells to suppress PPARgamma-mediated functions. Resveratrol inhibits cell growth of both Caco-2 and HCT-116 cells in a dose- and time-dependent manner (P < 0.001). In contrast to Caco-2-wild type cells (P < 0.05), resveratrol failed to increase SSAT activity in dominant-negative PPARgamma cells. PPARgamma involvement was further confirmed via ligand-dependent activation (P < 0.01) as well as by induction of cytokeratin 20 (P < 0.001) after resveratrol treatment. Coincubation with SB203580 abolished SSAT activation significantly in Caco-2 (P < 0.05) and HCT-116 (P < 0.01) cells. The involvement of p38 mitogen-activated protein kinase (MAPK) was further confirmed by a resveratrol-mediated phosphorylation of p38 protein in both cell lines. Resveratrol further increased the expression of PPARgamma coactivator PGC-1alpha (P < 0.05) as well as SIRT1 (P < 0.01) in a dose-dependent manner after 24 hours of incubation. Based on our findings, p38 MAPK and transcription factor PPARgamma can be considered as molecular targets of resveratrol in the regulation of cell proliferation and SSAT activity, respectively, in a cell culture model of colon cancer.

De novo ceramide biosynthesis is associated with resveratrol-induced inhibition of ornithine decarboxylase activity.

Previous studies could demonstrate, that the naturally occuring polyphenol resveratrol inhibits cell growth of colon carcinoma cells at least in part by inhibition of protooncogene ornithine decarboxylase (ODC). The objective of this study was to provide several lines of evidence suggesting that the induction of ceramide synthesis is involved in this regulatory mechanisms. Cell growth was determined by BrdU incorporation and crystal violet staining. Ceramide concentrations were detected by HPLC-coupled mass-spectrometry. Protein levels were examined by Western blot analysis. ODC activity was assayed radiometrically measuring [(14)CO(2)]-liberation. A dominant-negative PPARgamma mutant was transfected in Caco-2 cells to suppress PPARgamma-mediated functions. Antiproliferative effects of resveratrol closely correlate with a dose-dependent increase of endogenous ceramides (p<0.001). Compared to controls the cell-permeable ceramide analogues C2- and C6-ceramide significantly inhibit ODC-activity (p<0.001) in colorectal cancer cells. C6-ceramide further diminished protein levels of protooncogenes c-myc (p<0.05) and ODC (p<0.01), which is strictly related to the ability of ceramides to inhibit cell growth in a time- and dose-dependent manner. These results were further confirmed using inhibitors of sphingolipid metabolism, where only co-incubation with a serine palmitoyltransferase (SPT) inhibitor could significantly counteract resveratrol-mediated actions. These data suggest that the induction of ceramide de novo biosynthesis but not hydrolysis of sphingomyelin is involved in resveratrol-mediated inhibition of ODC. In contrast to the regulation of catabolic spermidine/spermine acetyltransferase by resveratrol, inhibitory effects on ODC occur PPARgamma-independently, indicating independent pathways of resveratrol-action. Due to our findings resveratrol could show great chemopreventive and therapeutic potential in the treatment of colorectal cancers.

Peroxisome proliferator-activated receptor alpha (PPAR alpha) down-regulation in cystic fibrosis lymphocytes.

BACKGROUND: PPARs exhibit anti-inflammatory capacities and are potential modulators of the inflammatory response. We hypothesized that their expression and/or function may be altered in cystic fibrosis (CF), a disorder characterized by an excessive host inflammatory response. METHODS: PPARalpha, beta and gamma mRNA levels were measured in peripheral blood cells of CF patients and healthy subjects via RT-PCR. PPARalpha protein expression and subcellular localization was determined via western blot and immunofluorescence, respectively. The activity of PPARalpha was analyzed by gel shift assay. RESULTS: In lymphocytes, the expression of PPARalpha mRNA, but not of PPARbeta, was reduced (-37%; p < 0.002) in CF patients compared with healthy persons and was therefore further analyzed. A similar reduction of PPARalpha was observed at protein level (-26%; p < 0.05). The transcription factor was mainly expressed in the cytosol of lymphocytes, with low expression in the nucleus. Moreover, DNA binding activity of the transcription factor was 36% less in lymphocytes of patients (p < 0.01). For PPARalpha and PPARbeta mRNA expression in monocytes and neutrophils, no significant differences were observed between CF patients and healthy persons. In all cells, PPARgamma mRNA levels were below the detection limit. CONCLUSION: Lymphocytes are important regulators of the inflammatory response by releasing cytokines and antibodies. The diminished lymphocytic expression and activity of PPARalpha may therefore contribute to the inflammatory processes that are observed in CF.

Restoration of E-cadherin sensitizes human melanoma cells for apoptosis.

Cell-cell adhesion is considered to be important in the development and maintenance of organ tissue. The spatial association between melanocytes and keratinocytes within human epidermis is achieved by homophilic interaction of E-cadherin molecules located on adjacent cells. In contrast, downregulation of E-cadherin expression in melanoma cells is considered as a key event in metastasis. Besides the adhesive properties, E-cadherin serves as a signal receptor linking to the cadherin-catenin signaling complex. As cadherins act as negative regulators of beta-catenin, a contribution to tumor formation seems likely. In the present study, it was tested whether ectopic expression of E-cadherin triggers apoptosis in human melanoma cell lines (G-361, JPC-298, SK-Mel-13). It was found that restoration of E-cadherin caused sensitization against drug-induced apoptosis. Particularly, the release of mitochondrial cytochrome c was increased in response to staurosporine. Moreover, activation of caspase-3 and caspase-8 was elevated. Similarly, DNA fragmentation, serving as a marker for advanced apoptosis, was amplified in cells transduced with E-cadherin. Interestingly, transduction with an E-cadherin construct lacking the extracellular domain showed no modified apoptosis. In conclusion, our findings suggest therapeutic strategies that enable expression of E-cadherin in order to sensitize human melanoma cells towards apoptosis.

Hypotonic stress induces E-cadherin expression in cultured human keratinocytes.

Human epidermis marks the interface between internal and external environments with the major task being to maintain body hydration. Alternating exposure of skin to a dry or humid environment is likely to cause changes in the epidermal water gradient resulting in osmotic alterations of epidermal keratinocytes. The present in vitro approach studied the effect of hypotonicity on cell-cell contact. It was demonstrated that hypotonic stress applied to human epithelial cells (HaCaT, A-431) induced upregulation of E-cadherin at both, the protein and mRNA level. 5'-deletional mutants of the E-cadherin promoter identified an element ranging from -53 to +31 that conveyed strong transactivation under hypotonic stress. In order to define relevant upstream regulators members of the MAP kinase family, the epidermal growth factor receptor (EGFR) and protein kinase B/Akt (PKB/Akt) were investigated. Hypotonic conditions led to a fast activation of ERK1/2, SAPK/JNK, p38, EGFR and PKB/Akt with distinct activation patterns. Experiments using specific inhibitors showed that p38 contributes to the E-cadherin transactivation under hypotonic conditions. Further upstream, adhesion was found to be a prerequisite for E-cadherin transactivation in this model. In summary, the present study provides evidence that E-cadherin is an osmo-sensitive gene that responds to hypotonic stress. The function of this regulation may be found in morphological changes induced by cell swelling. It is likely that induction of E-cadherin contributes to the stabilization between adjacent cells in order to withstand the physical forces induced by hypotonicity.

Detection of human beta defensin-1 and -2 by RT-competitive multiplex PCR.

Although, the human epithelium is constantly challenged by a broad spectrum of microorganisms, invasive infections are rather rare. Recent findings suggest the expression of antimicrobial peptides by skin cells in order to provide an innate defensive barrier. In particular, peptides of the beta-defensin family offer antimicrobial activity against different pathogens including bacteria and fungi. Within this peptide family, hBD-1 is rather constitutively expressed while hBD-2 and hBD-3 expression depends on environmental conditions. The present paper introduces RT-competitive multiplex PCR as a precise tool to detect hBD-1 and hBD-2 expression on the transcriptional level. The method makes use of co-amplification of synthetic competitors along with referring wildtype targets. Competitor- and wildtype-derived products differ in size allowing signal discrimination using agarose gel electrophoresis. Regulation of gene transcripts is evaluated by comparison of competitor and corresponding wildtype signals. It was found that primary human keratinocytes stimulated with Escherichia coli cells for 8 h offered an upregulation of hBD-2 to about 2,000 fold, while hBD-1 was only marginally regulated. RT-competitive multiplex PCR is a simple and accurate method that enables new insights into defensin regulation under physiological and pathophysiological conditions.

Cationic lipid-protamine-DNA (LPD) complexes for delivery of antisense c-myc oligonucleotides.

In the present study, cationic lipid-peptide-DNA-complexes (LPDs) consisting of AH-Chol-liposomes and protamine-phosphodiester-oligonucleotide-particles (proticles) were introduced as carriers for antisense therapy. The LPDs were physically characterized, and a possible mechanism for adsorption of oligonucleotides (ODNs) was suggested. An increase in stability of ODNs against DNase I and serum nuclease digestion by these carriers was demonstrated. The hydrodynamic diameter increased after incubation with FCS which could be attributed to a protein coating of the particle surface. However, in cell culture medium lower particle sizes of the complexes occurred. In an antisense c-myc in vitro model, the effect of LPDs was tested using U937 cells. The C-MYC level was reduced after treatment of these antisense ODN carrier complexes. Furthermore, no changes in target mRNA concentration of the treated cells was found by reverse transcription and competitive multiplex-PCR.

Ligation of the beta4 integrin triggers adhesion behavior of human keratinocytes by an "inside-out" mechanism.

Carcinogenesis is considered as a multistep process involving functional changes in the hemidesmosomal organization. In normal skin keratinocytes, expression of the alpha(6)beta(4) integrin is restricted to the proliferative basal layer and mediates stable adhesion to the underlying basement membrane. Observations in carcinoma cells show a functional and spatial dissociation of the alpha(6)beta(4) integrin from the hemidesmosomal complex, which stimulates cell migration and, therefore, may contribute to carcinoma invasion. We now have evaluated the adhesion behavior of epithelial cells at different stages of transformation in response to activation of the beta(4) integrin. It is demonstrated that ligation of the beta(4) integrin augmented adhesion of carcinoma and pre-carcinoma cells to non-modified plastic. In contrast, adhesion behavior of normal human keratinocytes was not influenced by ligation of the beta(4) integrin. In order to explain the mechanism of beta(4)-mediated adhesion, the hypothesis of an "inside-out" activation of integrins was tested. Evidence is given that for cells expressing the alpha(6)beta(4) integrin, ligation of the beta(4) integrin increased beta(1) integrin-mediated adhesion. Furthermore, ligation of the beta(4) integrin led to phosphorylation of PKB/Akt at both phosphorylation sites. Functional blocking of PKB/Akt by dominant-negative overexpression decreased cell adhesion in response to beta(4) integrin ligation. Taken together, the present data establish a link between the ligation of the beta(4) integrin and beta(1) integrin-mediated cell adhesion in carcinoma and pre-carcinoma cells. Hence, these findings provide further insight into the conversion processes during carcinogenesis and show the beta(4) integrin to be a key regulator of cellular adhesion.

Selective non-steroidal glucocorticoid receptor agonists attenuate inflammation but do not impair intestinal epithelial cell restitution in vitro.

INTRODUCTION: Despite the excellent anti-inflammatory and immunosuppressive action of glucocorticoids (GCs), their use for the treatment of inflammatory bowel disease (IBD) still carries significant risks in terms of frequently occurring severe side effects, such as the impairment of intestinal tissue repair. The recently-introduced selective glucocorticoid receptor (GR) agonists (SEGRAs) offer anti-inflammatory action comparable to that of common GCs, but with a reduced side effect profile. METHODS: The in vitro effects of the non-steroidal SEGRAs Compound A (CpdA) and ZK216348, were investigated in intestinal epithelial cells and compared to those of Dexamethasone (Dex). GR translocation was shown by immunfluorescence and Western blot analysis. Trans-repressive effects were studied by means of NF-κB/p65 activity and IL-8 levels, trans-activation potency by reporter gene assay. Flow cytometry was used to assess apoptosis of cells exposed to SEGRAs. The effects on IEC-6 and HaCaT cell restitution were determined using an in vitro wound healing model, cell proliferation by BrdU assay. In addition, influences on the TGF-ß- or EGF/ERK1/2/MAPK-pathway were evaluated by reporter gene assay, Western blot and qPCR analysis. RESULTS: Dex, CpdA and ZK216348 were found to be functional GR agonists. In terms of trans-repression, CpdA and ZK216348 effectively inhibited NF-κB activity and IL-8 secretion, but showed less trans-activation potency. Furthermore, unlike SEGRAs, Dex caused a dose-dependent inhibition of cell restitution with no effect on cell proliferation. These differences in epithelial restitution were TGF-ß-independent but Dex inhibited the EGF/ERK1/2/MAPK-pathway important for intestinal epithelial wound healing by induction of MKP-1 and Annexin-1 which was not affected by CpdA or ZK216348. CONCLUSION: Collectively, our results indicate that, while their anti-inflammatory activity is comparable to Dex, SEGRAs show fewer side effects with respect to wound healing. The fact that SEGRAs did not have a similar effect on cell restitution might be due to a different modulation of EGF/ERK1/2 MAPK signalling.

A glycerin hydrogel-based wound dressing prevents peristomal infections after percutaneous endoscopic gastrostomy (PEG): a prospective, randomized study.

BACKGROUND: Despite the use of prophylactic antibiotics, peristomal infection is the most common complication of percutaneous endoscopic gastrostomy (PEG). A new glycerin hydrogel (GHG) wound dressing has been proposed to possess more effective antimicrobial properties but has not been tested in a larger trial. The aim of the study was therefore to assess the superiority of GHG regarding the incidence of peristomal wound infections during a 30-day postprocedure follow-up. METHODS: Sixty-eight patients with cancer undergoing PEG were recruited from 1 university and 2 general hospitals between January 2007 and December 2008. Patients were randomized to group 1 (34 patients), which received GHG, or group 2 (34 patients), which received a traditional wound dressing. Dressing changes were done at day 1 and weeks 1, 2, and 4 (group 1) vs daily changes during week 1 and at weeks 2 and 4 (group 2). The PEG site was assessed by using 2 different infection scores. RESULTS: At the end of the first and second weeks, a statistically significant reduction of the mean infection scores was seen in patients with GHG wound dressings (first week: 1.64 ± 1.6 vs 3.12 ± 2.69, P < .008; second week: 1.37 ± 1.11 vs 2.53 ± 2.37, P < .02). After 7 days, wound reactions occurred in 14.7% in the GHG group vs 47.05% in the traditional group (p <0.005). The GHG wound dressing required 5 times less frequent dressing changes. CONCLUSION: The GHG wound dressing significantly reduces peristomal wound infections and is a convenient, cost-effective alternative for wound management following PEG.

Isothiocyanate sulforaphane inhibits protooncogenic ornithine decarboxylase activity in colorectal cancer cells via induction of the TGF-ß/Smad signaling pathway.

SCOPE: The objective of this study was to elucidate molecular mechanisms behind the antitumor activities of the isothiocyanate sulforaphane (SFN) in colorectal cancer cells. METHODS AND RESULTS: Cell growth was determined by BrdU incorporation and crystal violet staining. Protein levels were examined by Western blot analysis. Ornithine decarboxylase (ODC) activity was assayed radiometrically. Reverse transcriptase-PCR was used for measuring mRNA expression. For reporter gene assays plasmids were transfected into cells via lipofection and luciferase activity was measured luminometrically. Acetyl-histone H3 and H4 chromatin immunoprecipitation (ChIP) assays were performed followed by PCR with TGF-ß-receptor II promoter specific primers. We could show that SFN-mediated cell growth inhibition closely correlates with a dose-dependent reduction of protein expression and enzymatic activity of ODC. This effect seems to be due to reduced protein levels and transactivation activity of transcription factor c-myc, a direct regulator of ODC expression, as a consequence of SFN-induced TGF-ß/Smad signaling. The coherency of these results was further confirmed by using TGF-ß receptor kinase inhibitor SB431542, which largely abolishes inhibitory effects of SFN on both, ODC activity and cell growth. CONCLUSION: Since elevated ODC enzyme activity is associated with enhanced tumor development, SFN may be a dietary phytochemical with potential to prevent carcinogenesis.