TGF-beta 1 induces human alveolar epithelial to mesenchymal cell transition (EMT)

Background: Fibroblastic foci are characteristic features in lung parenchyma of patients with idiopathic pulmonary fibrosis (IPF). They comprise aggregates of mesenchymal cells which underlie sites of unresolved epithelial injury and are associated with progression of fibrosis. However, the cellular origins of these mesenchymal phenotypes remain unclear. We examined whether the potent fibrogenic cytokine TGF-β1 could induce epithelial mesenchymal transition (EMT) in the human alveolar epithelial cell line, A549, and investigated the signaling pathway of TGF-β1-mediated EMT. Methods: A549 cells were examined for evidence of EMT after treatment with TGF-β1. EMT was assessed by: morphology under phase-contrast microscopy; Western analysis of cell lysates for expression of mesenchymal phenotypic markers including fibronectin EDA (Fn-EDA), and expression of epithelial phenotypic markers including E-cadherin (E-cad). Markers of fibrogenesis, including collagens and connective tissue growth factor (CTGF) were also evaluated by measuring mRNA level using RT-PCR, and protein by immunofluorescence or Western blotting. Signaling pathways for EMT were characterized by Western analysis of cell lysates using monoclonal antibodies to detect phosphorylated Erk1/2 and Smad2 after TGF-β1 treatment in the presence or absence of MEK inhibitors. The role of Smad2 in TGF-β1-mediated EMT was investigated using siRNA. Results: The data showed that TGF-β1, but not TNF-α or IL-1β, induced A549 cells with an alveolar epithelial type II cell phenotype to undergo EMT in a time-and concentration-dependent manner. The process of EMT was accompanied by morphological alteration and expression of the fibroblast phenotypic markers Fn-EDA and vimentin, concomitant with a downregulation of the epithelial phenotype marker E-cad. Furthermore, cells that had undergone EMT showed enhanced expression of markers of fibrogenesis including collagens type I and III and CTGF. MMP-2 expression was also evidenced. TGF-β1-induced EMT occurred through phosphorylation of Smad2 and was inhibited by Smad2 gene silencing; MEK inhibitors failed to attenuate either EMT-associated Smad2 phosphorylation or the observed phenotypic changes. Conclusion: Our study shows that TGF-β1 induces A549 alveolar epithelial cells to undergo EMT via Smad2 activation. Our data support the concept of EMT in lung epithelial cells, and suggest the need for further studies to investigate the phenomenon

Use of a Cholera Rapid Diagnostic Test during a Mass Vaccination Campaign in Response to an Epidemic in Guinea, 2012

During the 2012 cholera outbreak in the Republic of Guinea, the Ministry of Health, supported by Médecins Sans Frontières - Operational Center Geneva, used the oral cholera vaccine Shanchol as a part of the emergency response. The rapid diagnostic test (RDT) Crystal VC, widely used during outbreaks, detects lipopolysaccharide antigens of Vibrio cholerae O1 and O139, both included in Shanchol. In the context of reactive use of a whole-cell cholera vaccine in a region where cholera cases have been reported, it is essential to know what proportion of vaccinated individuals would be reactive to the RDT and for how long after vaccination

Fast and Slow Effects of Medial Olivocochlear Efferent Activity in Humans

Background: The medial olivocochlear (MOC) pathway modulates basilar membrane motion and auditory nerve activity on both a fast (10–100 ms) and a slow (10–100 s) time scale in guinea pigs. The slow MOC modulation of cochlear activity is postulated to aide in protection against acoustic trauma. However in humans, the existence and functional roles of slow MOC effects remain unexplored. Methodology/Principal Findings: By employing contralateral noise at moderate to high levels (68 and 83 dB SPL) as an MOC reflex elicitor, and spontaneous otoacoustic emissions (SOAEs) as a non-invasive probe of the cochlea, we demonstrated MOC modulation of human cochlear output both on a fast and a slow time scale, analogous to the fast and slow MOC efferent effects observed on basilar membrane vibration and auditory nerve activity in guinea pigs. The magnitude of slow effects was minimal compared with that of fast effects. Consistent with basilar membrane and auditory nerve activity data, SOAE level was reduced by both fast and slow MOC effects, whereas SOAE frequency was elevated by fast and reduced by slow MOC effects. The magnitudes of fast and slow effects on SOAE level were positively correlated. Conclusions/Significance: Contralateral noise up to 83 dB SPL elicited minimal yet significant changes in both SOAE leve

Chronic Respiratory Aeroallergen Exposure in Mice Induces Epithelial-Mesenchymal Transition in the Large Airways

Chronic allergic asthma is characterized by Th2-polarized inflammation and leads to airway remodeling and fibrosis but the mechanisms involved are not clear. To determine whether epithelial-mesenchymal transition contributes to airway remodeling in asthma, we induced allergic airway inflammation in mice by intranasal administration of house dust mite (HDM) extract for up to 15 consecutive weeks. We report that respiratory exposure to HDM led to significant airway inflammation and thickening of the smooth muscle layer in the wall of the large airways. Transforming growth factor beta-1 (TGF-β1) levels increased in mouse airways while epithelial cells lost expression of E-cadherin and occludin and gained expression of the mesenchymal proteins vimentin, alpha-smooth muscle actin (α-SMA) and pro-collagen I. We also observed increased expression and nuclear translocation of Snail1, a transcriptional repressor of E-cadherin and a potent inducer of EMT, in the airway epithelial cells of HDM-exposed mice. Furthermore, fate-mapping studies revealed migration of airway epithelial cells into the sub-epithelial regions of the airway wall. These results show the contribution of EMT to airway remodeling in chronic asthma-like inflammation and suggest that Th2-polarized airway inflammation can trigger invasion of epithelial cells into the subepithelial regions of the airway wall where they contribute to fibrosis, demonstrating a previously unknown plasticity of the airway epithelium in allergic airway disease

Cancer-associated fibroblasts are positively correlated with metastatic potential of human gastric cancers

<p>Abstract</p> <p>Background</p> <p>The prognosis of gastric cancer patients is difficult to predict because of defects in establishing the surgical-pathological features. Cancer-associated fibroblasts (CAFs) have been found to play prominent role in promoting tumor growth, invasion and metastasis. Thus raises the hypothesis that the extent of CAFs prevalence may help to establish the prognosis of gastric cancer patients.</p> <p>Methods</p> <p>Immunochemistry and realtime-PCR experiments were carried out to compare the expression of proteins which are specific markers of CAFs or secreted by CAFs in the tumor and normal tissue specimens. The extent of CAFs' prevalence was graded according to immunochemical staining, and correlation was further analyzed between CAFs' prevalence and other tumor characteristics which may influence the prognosis of gastric cancer patients.</p> <p>Results</p> <p>Nearly 80 percent of normal gastric tissues were negative or weak positive for CAFs staining, while more than 60 percent of gastric cancer tissues were moderate or strong positive for CAFs staining. Realtime-PCR results also showed significant elevated expression of FAP, SDF-1 and TGF-β1 in gastric cancer tissues compared to normal gastric tissues. Further analysis showed that CAFs' prevalence was correlated with tumor size, depth of the tumor, lymph node metastasis, liver metastasis or peritoneum metastasis.</p> <p>Conclusions</p> <p>Reactive cancer associated fibroblasts (CAFs) were frequently accumulated in gastric cancer tissues, and the prevalence of CAFs was correlated with tumor size, depth of the tumor and tumor metastasis, thus give some supports for establishing the prognosis of the gastric cancer patients.</p

Intrinsic genetic characteristics determine tumor-modifying capacity of fibroblasts: matrix metalloproteinase-3 5A/5A genotype enhances breast cancer cell invasion

Background Stromal fibroblasts can contribute to tumor invasion through the release of matrix metalloproteinases (MMPs). Population studies have suggested that single nucleotide polymorphisms (SNPs) in MMP genes influence levels of expression and may be associated with breast cancer risk and with disease progression. This study directly examined the impact of MMP SNP genotype on the ability of host fibroblasts to promote tumor cell invasion. Methods Primary breast fibroblasts were isolated from patients with (n = 13) or without (n = 19) breast cancer, and their ability to promote breast cancer cell invasion was measured in in vitro invasion assays. Fibroblast invasion-promoting capacity (IPC) was analyzed in relation to donor type (tumor or non-tumor patient), MMP-1, MMP-3, and MMP-9 SNP genotype and MMP activity using independent samples t test and analysis of variance. All statistical tests were two-sided. Results Tumor-derived fibroblasts promoted higher levels of invasion than normal fibroblasts (p = 0.041). When IPC was related to genotype, higher levels of IPC were generated by tumor fibroblasts with the high-expressing MMP-3 5A/5A genotype compared with the 5A/6A and 6A/6A genotypes (p = 0.05 and 0.07, respectively), and this was associated with enhanced MMP-3 release. The functional importance of MMP-3 was demonstrated by enhanced invasion in the presence of recombinant MMP-3, whereas reduction occurred in the presence of a specific MMP-3 inhibitor. An inverse relationship was demonstrated between fibroblast IPC and the high-expressing MMP-1 genotype (p = 0.031), but no relationship was seen with MMP-9 SNP status. In contrast, normal fibroblasts showed no variation in IPC in relation to MMP genotype, with MMP-3 5A/5A fibroblasts exhibiting significantly lower levels of IPC than their tumor-derived counterparts (p = 0.04). Conclusion This study has shown that tumor-derived fibroblasts exhibit higher levels of IPC than normal fibroblasts and that the MMP-3 5A/5A genotype contributes to this through enhanced MMP-3 release. Despite a high-expressing genotype, normal fibroblasts do not exhibit higher IPC or enhanced MMP release. This suggests that more complex changes occur in tumor-derived fibroblasts, enabling full expression of the MMP SNP genotype and these possibly are epigenetic in nature. The results do suggest that, in women with breast cancer, a high-expressing MMP-3 genotype may promote tumor progression more effectively

Signal Transmission in the Auditory System

Contains table of contents for Section 3, an introduction and reports on five research projects.National Institutes of Health Grant R01-DC-00194National Institutes of Health Grant P01-DC-00119Charles S. Draper Laboratory Contract DL-H-496015National Institutes of Health Grant R01 DC00238National Institutes of Health Grant R01-DC02258National Institutes of Health Grant T32-DC00038National Institutes of Health Grant RO1 DC00235National Institutes of Health Grant P01-DC00361National Institutes of Health Contract N01-DC-6-210

Dynamic and influential interaction of cancer cells with normal epithelial cells in 3D culture

BACKGROUND: The cancer microenvironment has a strong impact on the growth and dynamics of cancer cells. Conventional 2D culture systems, however, do not reflect in vivo conditions, impeding detailed studies of cancer cell dynamics. This work aims to establish a method to reveal the interaction of cancer and normal epithelial cells using 3D time-lapse. METHODS: GFP-labelled breast cancer cells, MDA-MB-231, were co-cultured with mCherry-labelled non-cancerous epithelial cells, MDCK, in a gel matrix. In the 3D culture, the epithelial cells establish a spherical morphology (epithelial sphere) thus providing cancer cells with accessibility to the basal surface of epithelia, similar to the in vivo condition. Cell movement was monitored using time-lapse analyses. Ultrastructural, immunocytochemical and protein expression analyses were also performed following the time-lapse study. RESULTS: In contrast to the 2D culture system, whereby most MDA-MB-231 cells exhibit spindle-shaped morphology as single cells, in the 3D culture the MDA-MB-231 cells were found to be single cells or else formed aggregates, both of which were motile. The single MDA-MB-231 cells exhibited both round and spindle shapes, with dynamic changes from one shape to the other, visible within a matter of hours. When co-cultured with epithelial cells, the MDA-MB-231 cells displayed a strong attraction to the epithelial spheres, and proceeded to surround and engulf the epithelial cell mass. The surrounded epithelial cells were eventually destroyed, becoming debris, and were taken into the MDA-MB-231 cells. However, when there was a relatively large population of normal epithelial cells, the MDA-MB-231 cells did not engulf the epithelial spheres effectively, despite repeated contacts. MDA-MB-231 cells co-cultured with a large number of normal epithelial cells showed reduced expression of monocarboxylate transporter-1, suggesting a change in the cell metabolism. A decreased level of gelatin-digesting ability as well as reduced production of matrix metaroproteinase-2 was also observed. CONCLUSIONS: This culture method is a powerful technique to investigate cancer cell dynamics and cellular changes in response to the microenvironment. The method can be useful for various aspects such as; different combinations of cancer and non-cancer cell types, addressing the organ-specific affinity of cancer cells to host cells, and monitoring the cellular response to anti-cancer drugs. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12935-014-0108-6) contains supplementary material, which is available to authorized users

STAT3 can be activated through paracrine signaling in breast epithelial cells

<p>Abstract</p> <p>Background</p> <p>Many cancers, including breast cancer, have been identified with increased levels of phosphorylated or the active form of Signal Transducers and Activators of Transcription 3 (STAT3) protein. However, whether the tumor microenvironment plays a role in this activation is still poorly understood.</p> <p>Methods</p> <p>Conditioned media, which contains soluble factors from MDA-MB-231 and MDA-MB-468 breast cancer cells and breast cancer associated fibroblasts, was added to MCF-10A breast epithelial and MDA-MB-453 breast cancer cells. The stimulation of phosphorylated STAT3 (p-STAT3) levels by conditioned media was assayed by Western blot in the presence or absence of neutralized IL-6 antibody, or a JAK/STAT3 inhibitor, JSI-124. The stimulation of cell proliferation in MCF-10A cells by conditioned media in the presence or absence of JSI-124 was subjected to MTT analysis. IL-6, IL-10, and VEGF levels were determined by ELISA analysis.</p> <p>Results</p> <p>Our results demonstrated that conditioned media from cell lines with constitutively active STAT3 are sufficient to induce p-STAT3 levels in various recipients that do not possess elevated p-STAT3 levels. This signaling occurs through the JAK/STAT3 pathway, leading to STAT3 phosphorylation as early as 30 minutes and is persistent for at least 24 hours. ELISA analysis confirmed a correlation between elevated levels of IL-6 production and p-STAT3. Neutralization of the IL-6 ligand or gp130 was sufficient to block increased levels of p-STAT3 (Y705) in treated cells. Furthermore, soluble factors within the MDA-MB-231 conditioned media were also sufficient to stimulate an increase in IL-6 production from MCF-10A cells.</p> <p>Conclusion</p> <p>These results demonstrate STAT3 phosphorylation in breast epithelial cells can be stimulated by paracrine signaling through soluble factors from both breast cancer cells and breast cancer associated fibroblasts with elevated STAT3 phosphorylation. The induction of STAT3 phosphorylation is through the IL-6/JAK pathway and appears to be associated with cell proliferation. Understanding how IL-6 and other soluble factors may lead to STAT3 activation via the tumor microenvironment will provide new therapeutic regimens for breast carcinomas and other cancers with elevated p-STAT3 levels.</p