Molecular mechanisms in uterine epithelium during trophoblast binding: the role of small GTPase RhoA in human uterine Ishikawa cells

BACKGROUND: Embryo implantation requires that uterine epithelium develops competence to bind trophoblast to its apical (free) poles. This essential element of uterine receptivity seems to depend on a destabilisation of the apico-basal polarity of endometrial epithelium. Accordingly, a reorganisation of the actin cytoskeleton regulated by the small GTPase RhoA plays an important role in human uterine epithelial RL95-2 cells for binding of human trophoblastoid JAR cells. We now obtained new insight into trophoblast binding using human uterine epithelial Ishikawa cells. METHODS: Polarity of Ishikawa cells was investigated by electron microscopy, apical adhesiveness was tested by adhesion assay. Analyses of subcellular distribution of filamentous actin (F-actin) and RhoA in apical and basal cell poles were performed by confocal laser scanning microscopy (CLSM) with and without binding of JAR spheroids as well as with and without inhibition of small Rho GTPases by Clostridium difficile toxin A (toxin A). In the latter case, subcellular distribution of RhoA was additionally investigated by Western blotting. RESULTS: Ishikawa cells express apical adhesiveness for JAR spheroids and moderate apico-basal polarity. Without contact to JAR spheroids, significantly higher signalling intensities of F-actin and RhoA were found at the basal as compared to the apical poles in Ishikawa cells. RhoA was equally distributed between the membrane fraction and the cytosol fraction. Levels of F-actin and RhoA signals became equalised in the apical and basal regions upon contact to JAR spheroids. After inhibition of Rho GTPases, Ishikawa cells remained adhesive for JAR spheroids, the gradient of fluorescence signals of F-actin and RhoA was maintained while the amount of RhoA was reduced in the cytosolic fraction with a comparable increase in the membrane fraction. CONCLUSION: Ishikawa cells respond to JAR contact as well as to treatment with toxin A with rearrangement of F-actin and small GTPase RhoA but seem to be able to modify signalling pathways in a way not elucidated so far in endometrial cells. This ability may be linked to the degree of polar organisation observed in Ishikawa cells indicating an essential role of cell phenotype modification in apical adhesiveness of uterine epithelium for trophoblast in vivo

Efficacy of a Mycotoxin Binder against Dietary Fumonisin, Deoxynivalenol, and Zearalenone in Rats

It was hypothesized that a mycotoxin binder, Grainsure E, would inhibit adverse effects of a mixture of fumonisin B1, deoxynivalenol, and zearalenone in rats. For 14 and 28 days, 8–10 Sprague–Dawley rats were fed control diet, Grainsure E (0.5%), toxins (7 μg fumonisin B1/g, 8 μg of deoxynivalenol/g and 0.2 μg of zearalenone/g), toxins (12 μg of fumonisin B1/g, 9 μg of deoxynivalenol/g, and 0.2 μg of zearalenone/g + Grainsure E), or pair-fed to control for food intake of toxin-fed rats. After 28 days, decreased body weight gain was prevented by Grainsure E in toxin-fed female rats, indicating partial protection against deoxynivalenol and fumonisin B1. Two effects of fumonisin B1 were partly prevented by Grainsure E in toxin-fed rats, increased plasma alanine transaminase (ALT) and urinary sphinganine/sphingosine, but sphinganine/sphingosine increase was not prevented in females at the latter time point. Grainsure E prevented some effects of fumonisin B1 and deoxynivalenol in rats

The relationship between low back pain and leisure time physical activity in a working population of cleaners - a study with weekly follow-ups for 1 year

<p>Abstract</p> <p>Background</p> <p>Low back pain (LBP) and leisure time physical activity (LTPA) are considered to be closely related, and clinical guidelines for the treatment of acute LBP recommend patients stay physically active. However, the documentation for this recommendation is sparse and based on studies involving patient populations. The purpose of the study was (1) to investigate the correlation between LBP and LTPA on a weekly basis over the course of a year in a high-risk group of cleaners; and (2) to investigate if maintaining LTPA during an episode of acute LBP has a positive effect on LBP intensity in the subsequent 4 weeks.</p> <p>Methods</p> <p>188 cleaners consented to participate in a 52-week text message survey about hours of LTPA and intensity of LBP (from 0 to 9) over the previous 7 days. The correlation between LBP and LTPA was calculated by Pearson correlation coefficient. During an episode of acute LBP, a mixed effect logistic regression model was used to investigate whether cleaners who maintain LTPA have a lower pain intensity and higher probability of returning to initial pain intensity within the following four weeks compared with cleaners who decrease LTPA during acute LBP.</p> <p>Results</p> <p>The correlation between weekly LTPA and LBP data was negative, but numerically low (r = -0.069) and statistically insignificant (<it>p </it>= 0.08). Among the 82 cleaners experiencing at least one episode of acute LBP, those maintaining LTPA during an episode of acute LBP did not have a lower pain intensity (average LBP intensity difference between groups of 0.06; 95% confidence interval (95% CI) of -0.417 to 0.539) or higher probability of returning to initial pain level (Odds ratio 1,02; 95% CI of 0.50 to 2.09) in the following four weeks compared with cleaners decreasing LTPA during acute LBP.</p> <p>Conclusions</p> <p>Hours of LTPA and intensity of LBP measured on a weekly basis throughout a year showed no close correlation. Maintaining LTPA during an episode of acute LBP did not result in a positive effect on LBP in the following 4 weeks. Documentation of LTPA recommendations for acute LBP in working populations is still needed.</p

Chemotaxis of Cell Populations through Confined Spaces at Single-Cell Resolution

Cell migration is crucial for both physiological and pathological processes. Current in vitro cell motility assays suffer from various drawbacks, including insufficient temporal and/or optical resolution, or the failure to include a controlled chemotactic stimulus. Here, we address these limitations with a migration chamber that utilizes a self-sustaining chemotactic gradient to induce locomotion through confined environments that emulate physiological settings. Dynamic real-time analysis of both population-scale and single-cell movement are achieved at high resolution. Interior surfaces can be functionalized through adsorption of extracellular matrix components, and pharmacological agents can be administered to cells directly, or indirectly through the chemotactic reservoir. Direct comparison of multiple cell types can be achieved in a single enclosed system to compare inherent migratory potentials. Our novel microfluidic design is therefore a powerful tool for the study of cellular chemotaxis, and is suitable for a wide range of biological and biomedical applications

Genes targeted by the estrogen and progesterone receptors in the human endometrial cell lines HEC1A and RL95-2

<p>Abstract</p> <p>Background</p> <p>When the steroid hormones estrogen and progesterone bind to nuclear receptors, they have transcriptional impact on target genes in the human endometrium. These transcriptional changes have a critical function in preparing the endometrium for embryo implantation.</p> <p>Methods</p> <p>382 genes were selected, differentially expressed in the receptive endometrium, to study their responsiveness of estrogen and progesterone. The endometrial cell lines HEC1A and RL95-2 were used as experimental models for the non-receptive and receptive endometrium, respectively. Putative targets for activated steroid hormone receptors were investigated by chromatin immunoprecipitation (ChIP) using receptor-specific antibodies. Promoter occupancy of the selected genes by steroid receptors was detected in ChIP-purified DNA by quantitative PCR (qPCR). Expression analysis by reverse transcriptase (RT)-PCR was used to further investigate hormone dependent mRNA expression regulation of a subset of genes.</p> <p>Results</p> <p>ChIP-qPCR analysis demonstrated that each steroid hormone receptor had distinct group of target genes in the endometrial cell lines. After estradiol treatment, expression of estrogen receptor target genes predominated in HEC1A cells (n = 137) compared to RL95-2 cells (n = 35). In contrast, expression of progesterone receptor target genes was higher in RL95-2 cells (n = 83) than in HEC1A cells (n = 7) after progesterone treatment. RT-PCR analysis of 20 genes demonstrated transcriptional changes after estradiol or progesterone treatment of the cell lines.</p> <p>Conclusions</p> <p>Combined results from ChIP-qPCR and RT-PCR analysis showed different patterns of steroid hormone receptor occupancy at target genes, corresponding to activation or suppression of gene expression after hormone treatment of HEC1A and RL95-2 cell lines.</p

Short term culture of breast cancer tissues to study the activity of the anticancer drug taxol in an intact tumor environment

BACKGROUND: Sensitivity of breast tumors to anticancer drugs depends upon dynamic interactions between epithelial tumor cells and their microenvironment including stromal cells and extracellular matrix. To study drug-sensitivity within different compartments of an individual tumor ex vivo, culture models directly established from fresh tumor tissues are absolutely essential. METHODS: We prepared 0.2 mm thick tissue slices from freshly excised tumor samples and cultivated them individually in the presence or absence of taxol for 4 days. To visualize viability, cell death, and expression of surface molecules in different compartments of non-fixed primary breast cancer tissues we established a method based on confocal imaging using mitochondria- and DNA-selective dyes and fluorescent-conjugated antibodies. Proliferation and apoptosis was assessed by immunohistochemistry in sections from paraffin-embedded slices. Overall viability was also analyzed in homogenized tissue slices by a combined ATP/DNA quantification assay. RESULTS: We obtained a mean of 49 tissue slices from 22 breast cancer specimens allowing a wide range of experiments in each individual tumor. In our culture system, cells remained viable and proliferated for at least 4 days within their tissue environment. Viability of tissue slices decreased significantly in the presence of taxol in a dose-dependent manner. A three-color fluorescence viability assay enabled a rapid and authentic estimation of cell viability in the different tumor compartments within non-fixed tissue slices. CONCLUSION: We describe a tissue culture method combined with a novel read out system for both tissue cultivation and rapid assessment of drug efficacy together with the simultaneous identification of different cell types within non-fixed breast cancer tissues. This method has potential significance for studying tumor responses to anticancer drugs in the complex environment of a primary cancer tissue