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Background and Objective: Surrogate endpoints enable determination of meaningful treatment effects more efficiently than applying the endpoint of ultimate interest. We used data from trials of nintedanib in subjects with pulmonary fibrosis to assess decline in forced vital capacity (FVC) as a surrogate for mortality. Methods: Data from the nintedanib and placebo groups of trials in subjects with idiopathic pulmonary fibrosis, other forms of progressive pulmonary fibrosis, and pulmonary fibrosis due to systemic sclerosis (NCT00514683, NCT01335464, NCT01335477, NCT01979952, NCT02999178, NCT02597933) were pooled. Using joint models for longitudinal and time‐to‐event data, we assessed the association between decline in FVC % predicted and time to death over 52 weeks. The rate of change in FVC % predicted and the current value of FVC % predicted were modelled longitudinally and estimates applied as predictors in time‐to‐event models. Results: Among 2583 subjects with pulmonary fibrosis, both a greater rate of decline in FVC % predicted and a lower current value of FVC % predicted were associated with an increased risk of death over 52 weeks (HR 1.79 [95% CI: 1.57, 2.03] and HR 1.24 [1.17, 1.32] per 5‐percentage point decrease, respectively). Associations between the rate of change in FVC % predicted and the risk of death were consistent between patients with IPF and other ILDs. Conclusion: Data from clinical trials in subjects with pulmonary fibrosis of diverse aetiology demonstrate a strong association between decline in FVC % predicted and mortality over 52 weeks, supporting FVC decline as a surrogate for mortality in these patients. We used data from clinical trials of nintedanib in subjects with pulmonary fibrosis of diverse aetiology to demonstrate a strong association between decline in FVC per cent predicted and mortality over 52 weeks, supporting the use of FVC decline as a surrogate for mortality in clinical trials. [ABSTRACT FROM AUTHOR]

Objective: In the SENSCIS trial in subjects with systemic sclerosis–associated interstitial lung disease (SSc‐ILD), nintedanib reduced the rate of decline in forced vital capacity (FVC) over 52 weeks by 44% versus placebo. This study was undertaken to investigate the effects of nintedanib on categorical changes in FVC and other measures of ILD progression. Methods: In post hoc analyses, we assessed the proportions of subjects with categorical changes in FVC % predicted at week 52 and the time to absolute decline in FVC of ≥5% predicted or death and absolute decline in FVC of ≥10% predicted or death. Results: A total of 288 subjects received nintedanib and 288 subjects received placebo. At week 52, in subjects treated with nintedanib and placebo, respectively, 55.7% and 66.3% had any decline in FVC % predicted, 13.6% and 20.1% had a decline in FVC of >5% to ≤10% predicted, and 3.5% and 5.2% had a decline in FVC of >10% to ≤15% predicted; 34.5% and 43.8% had a decrease in FVC of ≥3.3% predicted (proposed minimal clinically important difference [MCID] for worsening of FVC), while 23.0% and 14.9% had an increase in FVC of ≥3.0% predicted (proposed MCID for improvement in FVC). Over 52 weeks, the hazard ratio (HR) for an absolute decline in FVC of ≥5% predicted or death with nintedanib versus placebo was 0.83 (95% confidence interval [95% CI] 0.66−1.06) (P = 0.14), and the HR for an absolute decline in FVC of ≥10% predicted was 0.64 (95% CI 0.43−0.95) (P = 0.029). Conclusion: These results suggest that nintedanib has a clinically relevant benefit on the progression of SSc‐ILD. [ABSTRACT FROM AUTHOR]

Objective: To assess the validity of patient self‐reported oral health measures as used in a large multi‐country survey for populations aged 50+. Background: Information on people's oral health status is important for assessing oral health needs within populations. However, clinical examination is not always possible. Patient self‐reported measures may provide an alternative when time and other resources are scarce. Materials and methods: Using oral health items from the Survey of Health, Ageing and Retirement in Europe (SHARE), self‐reported measures were collected from 186 patients receiving treatment at Heidelberg University Hospital. Self‐reports were compared with subsequent clinical examinations. Analyses were conducted for patients of all age groups and separately for patients aged 50+ (analogous to the SHARE study population). Diagnostic accuracy, agreement and correlation of patient‐reported information were examined using descriptive statistics and Bland‐Altman plots. Results: Patient‐reported presence or absence of a full tooth count was closely related to clinical measurement, both for all age groups (sensitivity: 93%; specificity: 92%) and persons aged 50+ (sensitivity: 100% specificity: 94%). Bland‐Altman plots indicate good agreement between patient‐ and clinical reports of the number of teeth at age 50+ (Concordance Correlation Coefficient = 0.95). Discriminatory power of patient‐reporting was good regarding presence vs absence of artificial teeth, but less robust regarding partial vs full replacement of missing teeth. Conclusion: Patient self‐evaluations provide reasonable estimates of clinical measures and appear sufficiently accurate for examining variations in the number of teeth, including among populations aged 50+. However, patient reports of the extent of replacement of missing teeth may not constitute reliable reflections of clinical conditions. [ABSTRACT FROM AUTHOR]

Previous studies have reported that repeat colonoscopies were often not conducted in the recommended time interval after screening colonoscopy. We prospectively followed participants of screening colonoscopy from Germany for 6 years to investigate utilization and determinants of repeat colonoscopies. In a longitudinal study on the effectiveness of screening colonoscopy in the state of Saarland (Germany), participants who had a screening colonoscopy between 2005 and 2007 were contacted by mail 6 years after screening and requested to fill in a standardized questionnaire on utilization of repeat colonoscopies. For all colonoscopies reported, colonoscopy and histology reports were requested from the physician(s). Of 6,407 screening participants, 2,212 (35%) have utilized another colonoscopy. Among participants with negative findings at screening (no adenomas), 962 (22%) had a subsequent colonoscopy within 6 years from screening, accounting for 43% of all patients with a repeat colonoscopy. Family history of CRC and detection of hyperplastic polyps were found to be determinants of higher repeat colonoscopy use. As many as 44% of the participants with low‐risk adenomas (N = 509) and 39% with high‐risk adenomas (N = 290) at screening did not utilize surveillance colonoscopy within 6 years. Utilization was better with higher school education, prior cancer screening participation and if high‐risk adenomas were detected, lower among current smokers and lowest among participants ≥70 years. New strategies will be required considering determinants of adherence to avoid unnecessary colonoscopies and to improve utilization of surveillance according to recommended time intervals among patients at higher risk of CRC in the future. What's new? Colonoscopy screening can detect and remove precursors of colorectal cancer but a surveillance colonoscopy is recommended to prevent recurrence. This study revealed major non‐adherence to existing guidelines for surveillance after screening colonoscopies in Germany: more than 40% of surveillance procedures were performed on patients with negative screening results while many patients with positive results did not utilize surveillance in the recommended time frame. The authors call for new strategies to improve utilization of follow‐up colonoscopies. [ABSTRACT FROM AUTHOR]

A recent randomized trial has suggested persisting protection from colorectal cancer (CRC) incidence and mortality of a single flexible sigmoidoscopy for up to 17 years and possibly beyond. We performed a simulation study to explore the time course and magnitude of protection provided by screening colonoscopy against CRC death over 25 years. Using data from the German national screening colonoscopy registry, a multistate Markov model was set up based on the adenoma–carcinoma pathway to estimate cumulative CRC mortality when different proportions of the population have a single screening colonoscopy at age 55, or two screening colonoscopies at ages 55 and 65. Cumulative CRC mortality continuously increased with age and reached 2.6 and 1.7% at age 80 in the absence of screening for men and women, respectively. A single colonoscopy at age 55, even with limited uptake, would lead to much lower cumulative mortality (0.7% for men and 0.5% for women at age 80 under 100% uptake). Relative mortality reduction continued to increase over more than 10 years and reached the maximum around 12–13 years after screening. Absolute risk reduction steadily increased throughout follow‐up and more than half of the total risk reduction would occur between 15–25 years. A repeat colonoscopy 10 years later further enhanced the effects and cumulative mortality remained at 0.1–0.2% under 100% uptake. Even a single (once‐only) screening colonoscopy has the potential to prevent most of CRC mortalities. Protective effects are expected to be long‐lasting and to become fully manifest after more than two decades from screening. What's new? Screening methods are only worthwhile if they actually increase survival, especially if they are expensive and labor intensive, such as colonoscopy. Yet observational studies only provide data for about 10 years of follow‐up. Here, the authors created a simulation to determine how long colonoscopy can prevent colon cancer deaths. Using data from the German national screening colonoscopy registry, they developed a model that estimates colon cancer mortality when different proportions of the population undergo colonoscopies. They found that the protective effect of even a single colonoscopy can last for 25 years, and repeat screenings can increase the protective effects. [ABSTRACT FROM AUTHOR]

Fecal immunochemical tests (FITs) for hemoglobin (Hb) are increasingly used for colorectal cancer (CRC) screening. We aimed to review, summarize and compare reported diagnostic performance of various FITs. PubMed and Web of Science were searched from inception to July 24, 2017. Data on diagnostic performance of quantitative FITs, conducted in colonoscopy‐controlled average‐risk screening populations, were extracted. Summary receiver operating characteristic (ROC) curves were plotted and correlations between thresholds, positivity rates (PRs), sensitivities and specificities were assessed. Seven test brands were investigated across 22 studies. Although reported sensitivities for CRC, advanced adenoma (AA) and any advanced neoplasm (AN) varied widely (ranges: 25–100%, 6–44% and 9–60%, respectively), with specificities for AN ranging from 82% to 99%, the estimates were very close to the respective summary ROC curves whose areas under the curve (95% CI) were 0.905 (0.88–0.94), 0.683 (0.67–0.70) and 0.710 (0.70–0.72) for CRC, AA and AN, respectively. The seemingly large heterogeneity essentially reflected variations in test thresholds (range: 2–82 µg Hb/g feces) and showed moderate correlations with sensitivity (r = −0.49) and specificity (r = 0.60) for AN. By contrast, observed PRs (range: 1–21%) almost perfectly correlated with sensitivity (r = 0.84) and specificity (r = −0.94) for AN. The apparent large heterogeneity in diagnostic performance between various FITs can be almost completely overcome by appropriate threshold adjustments. Instead of simply applying the threshold recommended by the manufacturer, screening programs should adjust the threshold to yield a desired PR which is a very good proxy indicator for the specificity and the subsequent colonoscopy workload. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: Dental diseases are the most common chronic diseases worldwide. Healthy teeth are vital for quality of life, particularly diet and nutrition. However, little information exists to inform health policymakers about potentially long‐lasting influences of early‐life conditions. The purpose of this study was to investigate the relation between early‐life socioeconomic conditions and number of natural teeth at age 50 and above. Methods: Analyses were conducted on cross‐sectional data from the Survey of Health, Ageing and Retirement in Europe (SHARE wave 5), which includes information on 41 560 respondents aged 50 years or older from 14 European countries and Israel. Using SHARE life history information, a series of regression models (OLS, Tobit) were estimated to analyse the relationship between socioeconomic conditions in earlier life and the number of teeth at age 50+. Results: Childhood socioeconomic background was associated with the number of natural teeth at age 50 and above, even after controlling for current determinants of oral health. Respondents who had had more than 25 books in their childhood household had a mean 1.4 (95% CI: 1.2‐1.5) more teeth than respondents with fewer books. Respondents who reported poor financial conditions during childhood had a mean 0.6 (95% CI: 0.3‐0.9) fewer teeth than respondents who reported better financial conditions in childhood. Conclusion: These findings substantiate the association between socioeconomic conditions in the early years of life and tooth retention to older adulthood and highlight the long‐lasting relation between childhood living conditions and oral health through the lifecourse. [ABSTRACT FROM AUTHOR]

Screening for colorectal cancer (CRC) is implemented in an increasing number of countries. We aimed to assess international variation in the prevalence of preclinical CRC and the resulting variation in positive and negative predictive values (PPVs, NPVs) of existing and potential CRC screening tests in various countries. Using age- and sex-specific CRC incidence data and transition rates from preclinical to clinical CRC we estimated overall and age- and sex-specific prevalence of preclinical CRC in the target population aged 50-74 years in different parts of the world. These prevalence estimates were used to derive PPVs and NPVs for existing and potential noninvasive screening tests with varying levels of sensitivity and specificity. Within all regions and countries, prevalence strongly increases with age and is higher in men than in women. In addition, major variation was seen between regions and countries, with overall prevalence varying between 1 and 0.1%. As a result, PPVs are expected to strongly vary between ∼10% for men in high incidence countries, such as Australia and Germany, and 1% for women in low incidence countries, whereas NPVs are expected to be consistently well above 99%. Variation in CRC prevalence profoundly affects expected PPVs of screening tests, and PPVs should be carefully considered when decisions on screening tests and strategies are made for specific populations and health care systems. Here, we provide estimates of preclinical CRC and expected PPVs and NPVs of noninvasive screening tests, which may enhance the empirical basis for planning of population-based CRC screening strategies. [ABSTRACT FROM AUTHOR]

Objectives Oral diseases are still among the most common chronic diseases globally with substantial detrimental impact especially on elderly people's health and well-being. However, limited evidence exists on international variation in the oral health status of the older population. We aimed to examine international variation in tooth loss and tooth replacement in the general population aged between 50 and 90 years. Methods A cross-sectional analysis of data from the fifth wave of the Survey of Health, Ageing and Retirement in Europe (SHARE) was conducted. The data cover 14 European countries (Austria, Belgium, Czech Republic, Denmark, Estonia, France, Germany, Italy, Luxembourg, the Netherlands, Slovenia, Spain, Sweden, and Switzerland) and Israel, and they were collected during the year 2013. Age-specific percentages of the population having all natural teeth, the age-specific numbers of natural (and artificial) teeth, and the age-specific percentages of full, partial, or no replacement of missing teeth were assessed with stratification by country. It was further evaluated to which extent proposed oral health goals concerning tooth loss at higher ages had been achieved. Results In total, 62 763 individuals were included in the study. Age-standardized mean numbers of natural teeth exhibited substantial variation, ranging from 14.3 (Estonia) to 24.5 (Sweden). The oral health goal of retaining at least 20 teeth at age 80 years was achieved by 25% of the population or less in most countries. A target concerning edentulism (≤15% in population aged 65-74 years) was reached in Sweden, Switzerland, Denmark, France, and Germany. Tooth replacement practices varied especially for a number of up to five missing teeth which were more likely to be replaced in Austria, Germany, Luxembourg, and Switzerland than in Israel, Denmark, Estonia, Spain, and Sweden. Conclusions This study suggests that the age-specific number of natural teeth and the practice of tooth replacement in the over 50s differ substantially among the included countries. The present results may be helpful in the formulation and evaluation of oral health goals in the older population. [ABSTRACT FROM AUTHOR]

Guaiac-based fecal occult blood tests (gFOBTs) are the most widely used noninvasive tests for colorectal cancer screening. While it is well known that they detect only a minority of colorectal adenomas, evidence for the characteristics of adenomas associated with detection is sparse. We derived estimates of the positive likelihood ratio (LR+), a summary measure of diagnostic performance, according to adenoma characteristics by comparing findings at colonoscopy among 19,208 and 181,128 participants who underwent colonoscopy to follow-up a positive gFOBT and as a primary screening examination, respectively, in Bavaria, Germany, in 2007-2009. Age and sex-adjusted estimates of LR+ (95% confidence intervals, 95% CI) ranged from 1.09 (1.05-1.13) for adenomas <1 cm to 2.52 (2.30-2.75) for adenomas >2 cm, and were much higher for pedunculated adenomas (1.96, 95% CI 1.85-2.08) than for flat or sessile adenomas (1.11, 95% CI 1.02-1.21 and 1.12, 95% CI 1.08-1.16, respectively). Villous or tubulovillous structure and dysplasia were likewise associated with a higher chance to be detected by gFOBT. Diagnostic performance was worse for proximal than for distal adenomas (age and sex adjusted LR+:1.16, 95% CI 1.09-1.23 and 1.35, 95% CI 1.29-1.41, respectively) which was explained by the lower proportions of large, pedunculated and nontubular adenomas in the proximal colon. Size, pedunculated shape, and nontubular histology are the key determinants of detection which also explain lower detection rates of adenomas located in the proximal colon. [ABSTRACT FROM AUTHOR]

Heparan sulfate 3- O-sulfotransferase 2 (HS3ST2), an enzyme mediating 3- O-sulfation of heparan sulfate (HS), is silenced by hypermethylation in breast cancer. As HS has an important co-receptor function for numerous signal transduction pathways, the phenotypical changes due to HS3ST2 reexpression were investigated in vitro using high and low invasive breast cancer cell lines. Compared to controls, highly invasive HS3ST2-expressing MDA-MB-231 cells showed enhanced Matrigel invasiveness, transendothelial migration and motility. Affymetrix screening and confirmatory real-time PCR and Western blotting analysis revealed increased expression of several matrix metalloproteinases, cadherin-11, E-cadherin and CEACAM-1, while protease inhibitor and annexin A10 expression were decreased. Low invasive HS3ST2 -expressing MCF-7 cells became even less invasive, with no change in gelatinolytic MMP activity. HS3ST2 expression increased HS-dependent basal and FGF2-specific signaling through the constitutively active p44/42 MAPK pathway in MDA-MB-231 cells. Increased MAPK activation was accompanied by upregulation of ß-catenin in MDA-MB-231, and of the transcription factor Tcf4 in both cell lines. Dysregulation of Tcf4-regulated ion transporters and increased cytosolic acidification were observed in HS3ST2-expressing MDA-MB-231 cells, which is a possible underlying cause of increased chemosensitivity towards doxorubicine and paclitaxel in these cells. This study provides the first in vitro evidence of the involvement of HS3ST2 in breast cancer cell invasion and chemosensitivity. [ABSTRACT FROM AUTHOR]

Guaiac-based fecal occult blood tests (gFOBTs) are the most commonly applied tests for colorectal cancer screening globally but have relatively poor sensitivity to detect colorectal neoplasms. Men have higher prevalences of colorectal neoplasms than women. In case of a positive gFOBT result, participants are referred to colonoscopy, independent of sex. To assess performance of gFOBT in routine screening practice, we assessed age and sex specific prevalences (age groups: 55-59, 60-64, 65-69 and 70-74) of colorectal neoplasms in 182,956 women and men undergoing colonoscopy for primary screening and in 20,884 women and men undergoing colonoscopy to follow-up a positive gFOBT in Bavaria, Germany, in 2007-2009. We conducted model calculations to estimate prevalences among gFOBT negative individuals. Analogous model calculations were performed for women and men tested positive or negative with fecal immunochemical tests. In all age groups (55-59, 60-64, 65-69 and 70-74 years), men undergoing colonoscopy for primary screening had substantially higher prevalences of any colorectal neoplasms and essentially the same prevalences of advanced colorectal neoplasms compared to women undergoing colonoscopy to follow-up a positive gFOBT. Model calculations suggest that men with negative gFOBT likewise have substantially higher prevalences of colorectal neoplasms than gFOBT positive women in each age group. Model calculations further indicate that no such sex paradoxon occurs, and a much clearer risk stratification can be achieved with fecal immunochemical tests. Our findings underline need to move forward from and overcome shortcomings of gFOBT-based colorectal cancer screening. [ABSTRACT FROM AUTHOR]

Many tumor cells are characterized by an increased net acid production. They extrude the excess protons mainly through the Na+/H+-exchanger NHE1. An increased NHE1 activity elevates the metastatic potential of tumor cells. Cell migration, a key step in the metastatic cascade, requires the formation and release of integrin-mediated cell-matrix contacts (focal adhesions). As NHE1 has been localized to focal adhesion sites, the present study tests the hypothesis that NHE1 generates measurable pH nanodomains right at focal adhesions. In order to ratiometrically measure pH close to the plasma membrane, we established a novel application of the total internal reflection fluorescence microscopy (TIRFM). Human melanoma cells were transfected with DsRed2-paxillin to identify focal adhesion sites. The pH-sensitive dyes BCECF and WGA-fluorescein were used to measure the submembranous cytosolic and the pericellular pH, respectively. Distinct pH nanodomains were found at focal adhesions, particularly at those located at the cell front, where NHE1 was concentrated. These sites featured a remarkably alkaline cytosolic and an acidic pericellular pH and thus a much steeper proton gradient across the plasma membrane compared to the rest of the cell. The generation of pH nanodomains could be assigned to NHE1-mediated H+ export because such pH domains could not be detected in NHE1-deficient cells. Given that both integrin avidity and mechanisms contributing to adhesion turnover are pH-sensitive, we propose that pH nanodomains at focal adhesions, locally created and maintained by NHE1 activity especially at the cell front, modulate adhesion dynamics in migrating cells. J. Cell. Physiol. 228: 1351-1358, 2013. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

microRNAs are small endogenous noncoding RNAs, which post-transcriptionally regulate gene expression. In breast cancer, overexpression of the transmembrane heparan sulfate proteoglycan syndecan-1, a predicted target of the oncomiR miR-10b, correlates with poor clinical outcome. To investigate the potential functional relationship of miR-10b and syndecan-1, MDA-MB-231 and MCF-7 breast cancer cells were transiently transfected with pre-miR-10b, syndecan-1 siRNA or control reagents, respectively. Altered cell behavior was monitored by proliferation, migration and invasion chamber assays, and time-lapse video microscopy. miR-10b overexpression induced post-transcriptional downregulation of syndecan-1, as demonstrated by quantitative real-time PCR (qPCR), flow cytometry, and 3′UTR luciferase assays, resulting in increased cancer cell migration and matrigel invasiveness. Syndecan-1 silencing generated a copy of this phenotype. Adhesion to fibronectin and laminin and basal cell proliferation was increased. Syndecan-1 coimmunoprecipitated with focal adhesion kinase, which showed increased activation upon syndecan-1 depletion. Affymetrix screening and confirmatory qPCR and Western blotting analysis of syndecan-1-deficient cells revealed upregulation of ATF-2, COX-2, cadherin-11, vinculin, actin γ 2, MYL9, transgelin-1, RhoA/C, matrix metalloproteinase 2 (MMP2) and heparanase, and downregulation of AML1/RUNX1, E-cadherin, CLDN1, p21WAF/CIP, cyclin-dependent kinase 6, TLR-4, PAI1/2, Collagen1alpha1, JHDM1D, Mpp4, MMP9, matrilin-2 and ANXA3/A10. Video microscopy demonstrated massively increased Rho kinase-dependent motility of syndecan-1-depleted cells, which displayed increased filopodia formation. We conclude that syndecan-1 is a novel target of the oncomiR miR-10b. Rho-GTPase-dependent modulation of cytoskeletal function and downregulation of E-cadherin expression are identified as relevant effectors of the miR-10b-syndecan-1 axis, which emerges as a promising target for the development of new therapeutic approaches for breast cancer. [ABSTRACT FROM AUTHOR]

Both molybdenum(VI) dichloride dioxide (MoO2Cl2) and its dimethylformamide (DMF) complex catalyze the addition of alcohols to isocyanates giving carbamates. Most additions proceeded to completion at room temperature within 20 min using as little as 0.1 mol% of the catalyst when working on a 1-mmol scale or just 100 ppm working on a 20-mmol scale. Sterically encumbered substrates reacted to completion when 1 mol% of the catalyst was employed. Diols, triols, and tetraols reacted with monoisocyanates likewise, as did monofunctional alcohols and diisocyanates. These pairings furnished di-, tri-, tetra-, and dicarbamates, respectively. Reactants, which were poorly soluble in CH2Cl2 at room temperature required elevating the temperature and possibly choosing a higher-boiling solvent (ClCH2CH2Cl, DMF) as well. Additions of diols to diisocyanates were feasible, too, giving polycarbamates as we presume. [ABSTRACT FROM AUTHOR]

Calcium-sensitive potassium channels (KCa3.1) are expressed in virtually all migrating cells. Their activity is required for optimal cell migration so that their blockade leads to slowing down. KCa3.1 channels must be inserted into the plasma membrane in order to elicit their physiological function. However, the plasma membrane of migrating cells is subject to rapid recycling by means of endo- and exocytosis. Here, we focussed on the endocytic internalization and the intracellular transport of the human isoform hKCa3.1. A hKCa3.1 channel construct with an HA-tag in the extracellularly located S3-S4 linker was transfected into migrating transformed renal epithelial MDCK-F cells. Channel internalization was visualized and quantified with immunofluorescence and a cell-based ELISA. Movement of hKCa3.1 channel containing vesicles as well as migration of MDCK-F cells were monitored by means of time lapse video microscopy. hKCa3.1 channels are endocytosed during migration. Most of the hKCa3.1 channel containing vesicles are moving at a speed of up to 2 µm/sec in a microtubule-dependent manner towards the front of MDCK-F cells. Our experiments indicate that endocytosis of hKCa3.1 channels is clathrin-dependent since they colocalize with clathrin adaptor proteins and since it is impaired when a C-terminal dileucine motif is mutated. The C-terminal dileucine motif is also important for the subcellular localization of hKCa3.1 channels in migrating cells. Mutated channels are no longer concentrated at the leading edge. We therefore propose that recycling of hKCa3.1 channels contributes to their characteristic subcellular distribution in migrating cells. J. Cell. Physiol. 227: 686-696, 2012. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

In vivo, cells are embedded in an environment generated and maintained by multiple cell-cell and cell-matrix interactions. While transiting the dermis metastasizing melanoma cells interact with the extracellular matrix (ECM) and fibroblasts. To study the roles of ECM components and fibroblasts in melanoma (B16V) cell migration and invasion, we established a co-culture system consisting of fibroblasts, their collagen-rich matrix and B16V cells. The crosstalk between B16V cells and fibroblasts was indicated by a clear increase in release and activity of matrix-metallo-protease-2. Time-lapse microscopy revealed that in B16V cells exposed to either decorin or chondroitin sulfates migration and invasion decreased by more than 50%. Decorin led to a reversible, chondroitin-6-sulfate to an irreversible, cytosolic acidification of B16V cells. Interestingly, decorin lowered NHE1 activity whereas chondroitin-6-sulfate did not. Furthermore, decorin and chondroitin-6-sulfate also acidified the pH at the cell surface which might prevent migration due to strong adhesion. In conclusion, the present co-culture system is an appropriate tool to analyze migration, invasion, and MMP release depending on cell-matrix interactions and the crosstalk between the invasive cells and those surrounded by their self-made matrix. We show a so far unknown function of decorin and chondroitin-6-sulfate: their ability to inhibit B16V cell migration by intracellular acidification. J. Cell. Physiol. 226: 2641-2650, 2011. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Migration and morphology of human melanoma cells (MV3) depend on extracellular pH (pHe) and the activity of the Na+/H+ exchanger NHE1. To distinguish effects of NHE1 activity per se from effects of pHe we compared an NHE1-deficient mutant with rescued and wild-type cells. Time lapse video microscopy was used to investigate migratory and morphological effects caused by pHe and NHE1 activity, and a membrane-bound fluorescein conjugate was employed for ratiometric pH measurements at the outer leaflet of the cell membrane. As long as NHE1 remained inactive due to deficiency or inhibition by cariporide (HOE642) neither migration nor morphology was affected by changes in pHe. Under these conditions pH at the outer leaflet of the plasma membrane was uniform all over the cell surface. The typical pH dependence of MV3 cell migration and morphology could be reconstituted by restoring NHE1 activity. At the same time the proton gradient at the outer leaflet of the plasma membrane with the higher proton concentration at the leading edge and the lower one at the cell rear was re-established as well. Hence, NHE1 activity generates a proton gradient at the cell surface accompanied by the cells' ability to respond to changes in pHe (bulk pH). We conclude that NHE1 activity contributes to the generation of a well-defined cell surface pH by creating a proton gradient at the outer leaflet of the plasma membrane that is needed for (i) the development of a variety of morphologies including a distinct polarity and (ii) migration. A missing proton gradient at the cell surface cannot be compensated for by varying pHe. [ABSTRACT FROM AUTHOR]

Cell migration is crucial for wound healing, immune defense, or formation of tumor metastases. In addition to the cytoskeleton, Ca2+ sensitive K+ channels (IK1) are also part of the cellular “migration machinery.” We showed that Ca2+ sensitive K+ channels support the retraction of the rear part of migrating MDCK-F cells by inducing a localized shrinkage at this cell pole. So far the molecular nature and in particular the subcellular distribution of these channels in MDCK-F cells is unknown. We compared the effect of IK1 channel blockers and activators on the current of a cloned IK1 channel from MDCK-F cells (cIK1) and the migratory behavior of these cells. Using IK1 channels labeled with a HA-tag or the enhanced green fluorescent protein we studied the subcellular distribution of the canine (cIK1) and the human (hIK1) channel protein in different migrating cells. The functional impact of cIK1 channel activity at the front or rear part of MDCK-F cells was assessed with a local superfusion technique and a detailed morphometric analysis. We show that it is cIK1 whose activity is required for migration of MDCK-F cells. IK1 channels are found in the entire plasma membrane, but they are concentrated at the cell front. This is in part due to membrane ruffling at this cell pole. However, there appears to be only little cIK1 channel activity at the front of MDCK-F cells. In our view this apparent discrepancy can be explained by differential regulation of IK1 channels at the front and rear part of migrating cells. © 2005 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Their glycolytic metabolism imposes an increased acid load upon tumour cells. The surplus protons are extruded by the Na+/H+ exchanger (NHE) which causes an extracellular acidification. It is not yet known by what mechanism extracellular pH (pHe) and NHE activity affect tumour cell migration and thus metastasis. We studied the impact of pHe and NHE activity on the motility of human melanoma (MV3) cells. Cells were seeded on/in collagen I matrices. Migration was monitored employing time lapse video microscopy and then quantified as the movement of the cell centre. Intracellular pH (pHi) was measured fluorometrically. Cell–matrix interactions were tested in cell adhesion assays and by the displacement of microbeads inside a collagen matrix. Migration depended on the integrin α2β1. Cells reached their maximum motility at pHe∼7.0. They hardly migrated at pHe 6.6 or 7.5, when NHE was inhibited, or when NHE activity was stimulated by loading cells with propionic acid. These procedures also caused characteristic changes in cell morphology and pHi. The changes in pHi, however, did not account for the changes in morphology and migratory behaviour. Migration and morphology more likely correlate with the strength of cell–matrix interactions. Adhesion was the strongest at pHe 6.6. It weakened at basic pHe, upon NHE inhibition, or upon blockage of the integrin α2β1. We propose that pHe and NHE activity affect migration of human melanoma cells by modulating cell–matrix interactions. Migration is hindered when the interaction is too strong (acidic pHe) or too weak (alkaline pHe or NHE inhibition). [ABSTRACT FROM AUTHOR]

Migration of microglial cells towards damaged tissue plays a key role in central nervous system regeneration under pathological conditions. Using time lapse video microscopy we show that lysophosphatidic acid (LPA) enhances chemokinetic migration of murine microglial cells. In the presence of 1 µ m LPA, the mean migration rate of microglial cells was increased 3.8-fold. In patch-clamp studies we demonstrate that LPA induces activation of a Ca2+-activated K+ current. Microglial Ca2+-activated K+ currents were abolished by either 50 n m charybdotoxin or 10 µ m clotrimazole. In contrast, 5 µ m paxilline did not have any significant effects on Ca2+-activated K+ currents. The LPA-stimulated migration of microglial cells was inhibited by blockers of IKCa1 Ca2+-activated K+ channels. The mean migration rate of LPA-stimulated cells was decreased by 61% in the presence of 50 n m charybdotoxin or by 51% during exposure to 10 µ m clotrimazole. Microglial migration was not inhibited by 5 µ m paxilline. It is concluded that IKCa1 Ca2+-activated K+ channels are required for LPA-stimulated migration of microglial cells. [ABSTRACT FROM AUTHOR]

Abstract Migration of microglial cells towards damaged tissue plays a key role in central nervous system regeneration under pathological conditions. Using time lapse video microscopy we show that lysophosphatidic acid (LPA) enhances chemokinetic migration of murine microglial cells. In the presence of 1 micro m LPA, the mean migration rate of microglial cells was increased 3.8-fold. In patch-clamp studies we demonstrate that LPA induces activation of a Ca(2+)-activated K(+) current. Microglial Ca(2+)-activated K(+) currents were abolished by either 50 nm charybdotoxin or 10 micro m clotrimazole. In contrast, 5 micro m paxilline did not have any significant effects on Ca(2+)-activated K(+) currents. The LPA-stimulated migration of microglial cells was inhibited by blockers of IKCa1 Ca(2+)-activated K(+) channels. The mean migration rate of LPA-stimulated cells was decreased by 61% in the presence of 50 nm charybdotoxin or by 51% during exposure to 10 micro m clotrimazole. Microglial migration was not inhibited by 5 micro m paxilline. It is concluded that IKCa1 Ca(2+)-activated K(+) channels are required for LPA-stimulated migration of microglial cells.