Autophagic activity in BC3H1 cells exposed to yessotoxin.

The marine toxin yessotoxin (YTX) can induce programmed cell death through both caspase-dependent and -independent pathways in various cellular systems. It appears to stimulate different forms of cellular stress causing instability among cell death mechanisms and making them overlap and cross-talk. Autophagy is one of the key pathways that can be stimulated by multiple forms of cellular stress which may determine cell survival or death. The present work evaluates a plausible link between ribotoxic stress and autophagic activity in BC3H1 cells treated with YTX. Such treatment produces massive cytoplasmic compartments as well as double-membrane vesicles termed autophagosomes which are typically observed in cells undergoing autophagy. The observed autophagosomes contain a large amount of ribosomes associated with the endoplasmic reticulum (ER). Western blotting analysis of Atg proteins and detection of the autophagic markers LC3-II and SQSTM1/p62 by flow cytometry and immunofluorescence verified autophagic activity during YTX-treatment. The present work supports the idea that autophagic activity upon YTX exposure may represent a response to ribotoxic stress.

Effects of the non-commensal Methylococcus capsulatus Bath on mammalian immune cells.

Dietary inclusions of a bacterial meal consisting mainly of the non-commensal, methanotrophic bacteria Methylococcus capsulatus Bath have been shown to ameliorate symptoms of intestinal inflammation in different animal models. In order to investigate the molecular mechanisms causing these effects, we have studied the influence of this strain on different immune cells central for the regulation of inflammatory responses. Effects were compared to those induced by the closely related strain M. capsulatus Texas and the well-described probiotic strain Escherichia coli Nissle 1917. M. capsulatus Bath induced macrophage polarization toward a pro-inflammatory phenotype, but not to the extent observed after exposure to E. coli Nissle 1917. Likewise, dose-dependent abilities to activate NF-κB transcription in U937 cells were observed, with E. coli Nissle 1917 being most potent. High levels of CD141 on human primary monocyte-derived dendritic cells (moDCs) were only detected after exposure to E. coli Nissle 1917, which collectively indicate a superior capacity to induce Th1 cell responses for this strain. On the other hand, the M. capsulatus strains were more potent in increasing the expression of the maturation markers CD80, CD83 and CD86 than E. coli Nissle 1917. M. capsulatus Bath induced the highest levels of IL-6, IL-10 and IL-12 secretion from dendritic cells, suggesting that this strain generally the post potent inducer of cytokine secretion. These results show that M. capsulatus Bath exhibit immunogenic properties in mammalian in vitro systems which diverge from that of E. coli Nissle 1917. This may provide clues to how M. capsulatus Bath influence the adaptive immune system in vivo. However, further in vivo experiments are required for a complete understanding of how this strain ameliorates intestinal inflammation in animal models.

In vitro comparison of the effects of probiotic, commensal and pathogenic strains on macrophage polarization.

Macrophages are important with respect to both innate and adaptive immune responses and are known to differentiate into pro-inflammatory M1- or anti-inflammatory M2-phenotypes following activation. In order to study how different bacteria affect macrophage polarization, we exposed murine RAW 264.7 macrophages to sixteen different strains representing probiotic strains, pathogens, commensals and strains of food origin. Increased inducible nitric oxide synthase (iNOS) or arginase-1 gene expression indicates M1 or M2 polarization, respectively, and was quantified by qRT-PCR. Strains of Escherichia and Salmonella elevated iNOS expression more so than strains of Enterococcus, Lactobacillus and Lactococcus, indicating that Gram-negative strains are more potent M1 inducers. However, strain-specific responses were observed. For instance, Escherichia coli Nissle 1917 was a poor inducer of iNOS gene expression compared to the other E. coli strains, while Enterococcus faecalis Symbioflor-1 was more potent in this respect compared to all the eleven Gram-positive strains tested. Macrophage polarization was further characterized by quantifying secreted pro- and anti-inflammatory cytokines. Exposure to the pathogen E. coli 042 produced a cytokine profile indicating M1 differentiation, which is in accordance with the PCR data. However, exposure to most strains resulted in either high or low secretion levels of all cytokines tested, rather than a clear M1 or M2 profile. In general, the Gram-negative strains induced high levels of cytokine secretion compared to the Gram-positive strains. Interestingly, strains of human origin had a higher impact on macrophages compared to strains of food origin.

The endothelin axis in the metastatic process of colon carcinoma.

BACKGROUND: The endothelin axis has recently emerged as an important factor in tumour metastasis. The aim of this study was to investigate the endothelin axis and its downstream pathways related to metastasis in colon carcinoma. MATERIALS AND METHODS: mRNA expression of 36 genes associated with the endothelin axis in 18 non-metastatic and 20 metastatic colon carcinomas with individual-matched normal mucosa were evaluated using real-time reverse transcription polymerase chain reaction. RESULTS: Seventeen out of 36 genes, including endothelin A receptor, were significantly overexpressed in the tumour tissue compared to the individual-matched normal mucosa. Seven out of 36 genes, including endothelin B receptor, were significantly down-regulated in tumour tissue. Phosphatase and tensin homolog (PTEN) was significantly down-regulated in the metastatic patients compared to the non-metastatic patients. CONCLUSION: This study indicated that central genes in the endothelin axis are overexpressed when colon tissue becomes malignant. Down-regulation of PTEN may promote a progressive phenotype of colon carcinomas.

Determination of thromboxanes, leukotrienes and lipoxins using high-temperature capillary liquid chromatography-tandem mass spectrometry and on-line sample preparation.

An on-line strong cation-exchange (SCX)-reversed-phase (RP) capillary liquid chromatographic (cLC) method with ion-trap tandem mass spectrometric (IT-MS/MS) detection for the simultaneous determination of thromboxane (TX) B(2), TXB(3), leukotriene (LT) B(4), LTD(4) and lipoxin (LX) A(4) in cell culture supernatants was developed and validated. In the present method, a high temperature (70 degrees C) was used for the separation on the analytical column to obtain efficient chromatography of the thromboxanes. An on-line sample preparation was performed, where peptides/proteins contained in the matrix were removed by the SCX column. Sample pre-treatment included dilution and filtration, and the analysis time including all sample preparation steps was 60min per sample. Limits of detection in the range of 1-4ng/mL cell culture supernatant, recoveries between 30% and 100%, within day precisions of less than 20% RSD and between day precisions of less than 30% RSD were obtained. Human mesenchymal stem cells (hMSCs) were stimulated with cytokine-containing supernatants derived from activated human T lymphocytes, and thromboxane, leukotriene and lipoxin production was analysed using the developed method. TXB(2) was found in cultures from both non-differentiated and differentiated hMSCs that were stimulated with a cytokine-containing supernatant obtained from activated T-cells.

Detecting pM concentrations of prostaglandins in cell culture supernatants by capillary SCX-LC-MS/MS.

A highly sensitive, improved online strong cation exchange (SCX)--RP capillary liquid chromatographic (cLC) method with IT mass spectrometric (IT-MS/MS) detection for the simultaneous determination of prostaglandin (PG)A(1), PGD(2), PGE(1), PGE(2), PGF(2alpha), 8-iso-(8i)PGF(2alpha), 6-keto-(6k)PGF(1alpha), and 15-Delta(12, 14)-deoxy-PGJ(2) (15dPGJ(2)) in cell culture supernatants was developed and validated. Pretreatment of the cell culture supernatants included only dilution and filtration, and the analysis time including all sample preparation steps was 60 min per sample. Peptides/proteins contained in the matrix were removed by the SCX column. LODs in the range of 8-44 pg/mL (25-120 pM) cell culture supernatant were obtained. Excellent linearity (R(2) > 0.99) and satisfactory recoveries and within- and between-day precisions were obtained. Human mesenchymal stem cells (hMSCs) were stimulated with tumor necrosis factor alpha (TNFalpha) or TNFalpha/IL-17, and PG production was analyzed using the developed method. The four PGs, 6kPGF(1a), PGF(1a), PGE(2), and PGE(1 )were detected both in nonstimulated and stimulated cells. The amount of PG produced by the cell increased when the cell was stimulated.

Human mesenchymal stem cell proliferation is regulated by PGE2 through differential activation of cAMP-dependent protein kinase isoforms.

The conditions used for in vitro differentiation of hMSCs contain substances that affect the activity and expression of cyclooxygenase enzymes (COX1/COX2) and thereby the synthesis of prostanoids. hMSC constitutively produce PGE2 when cultivated in vitro. In this study we have investigated effects of PGE2 on proliferation of hMSC. We here demonstrate that one of the main control molecules in the Wnt pathway, GSK-3 beta, is phosphorylated at the negative regulatory site ser-9 after treating the cells with PGE2. This phosphorylation is mediated by elevation of cAMP and subsequent activation of PKA. Furthermore, PGE2 treatment leads to enhanced nuclear translocation of beta-catenin, thus influencing cell proliferation. The presence of two PKA isoforms, types I and II, prompted us to investigate their individual contribution in PGE2-mediated regulation of proliferation. Specific activation of PKA type II with synthetic cAMP analogues, resulted in enhancement of proliferation. On the other side, we found that treatment of hMSC with high concentrations of PGE2 inhibited cell proliferation by arresting the cells in G0/G1 phase, an effect we found to be mediated by PKA I. Hence, the two different PKA isoforms seem to have opposing functions in the regulation of proliferation and differentiation in these cells.