The microfluidic lighthouse: an omnidirectional gradient generator.

Studies of chemotactic cell migration rely heavily on various assay systems designed to evaluate the ability of cells to move in response to attractant molecules. In particular, the development of microfluidics-based devices in recent years has made it possible to spatially distribute attractant molecules in graded profiles that are sufficiently stable and precise to test theoretical predictions regarding the accuracy and efficiency of chemotaxis and the underlying mechanism of stimulus perception. However, because the gradient is fixed in a direction orthogonal to the laminar flow and thus the chamber geometry, conventional devices are limited for the study of cell re-orientation to gradients that move or change directions. Here, we describe the development of a simple radially symmetric microfluidics device that can deliver laminar flow in 360°. A stimulant introduced either from the central inlet or by photo uncaging is focused into the laminar flow in a direction determined by the relative rate of regulated flow from multiple side channels. Schemes for flow regulation and an extended duplexed device were designed to generate and move gradients in desired orientations and speed, and then tested to steer cell migration of Dictyostelium and neutrophil-like HL60 cells. The device provided a high degree of freedom in the positioning and orientation of attractant gradients, and thus may serve as a versatile platform for studying cell migration, re-orientation, and steering.

B-RAF mutation and accumulated gene methylation in aberrant crypt foci (ACF), sessile serrated adenoma/polyp (SSA/P) and cancer in SSA/P.

BACKGROUND: Sessile serrated adenomas/polyps (SSA/Ps) are a putative precursor of colon cancer with microsatellite instability (MSI). However, the developmental mechanism of SSA/P remains unknown. We performed genetic analysis and genome-wide DNA methylation analysis in aberrant crypt foci (ACF), SSA/P, and cancer in SSA/P specimens to show a close association between ACF and the SSA/P-cancer sequence. We also evaluated the prevalence and number of ACF in SSA/P patients. METHODS: ACF in the right-side colon were observed in 36 patients with SSA/Ps alone, 2 with cancers in SSA/P, and 20 normal subjects and biopsied under magnifying endoscopy. B-RAF mutation and MSI were analysed by PCR-restriction fragment length polymorphism (RFLP) and PCR-SSCP, respectively, in 15 ACF, 20 SSA/P, and 2 cancer specimens. DNA methylation array analysis of seven ACF, seven SSA/P, and two cancer in SSA/P specimens was performed using the microarray-based integrated analysis of methylation by isochizomers (MIAMI) method. RESULTS: B-RAF mutations were frequently detected in ACF, SSA/P, and cancer in SSA/P tissues. The number of methylated genes increased significantly in the order of ACF

IL-22 produced by cancer-associated fibroblasts promotes gastric cancer cell invasion via STAT3 and ERK signaling.

BACKGROUND: Interleukin-22 (IL-22) has been recently highlighted owing to its biological significance in the modulation of tissue responses during inflammation. However, the role of IL-22 in carcinogenesis has remained unclear. Here, we investigated the pathophysiological significance of IL-22 expression in gastric cancer tissues and examined the mechanism by which IL-22 promotes gastric cancer cell invasion. METHODS: Human gastric cancer specimens were analysed by immunohistochemistry for expression of IL-22 and IL-22 receptor 1 (IL-22R1). The effects of IL-22-induced STAT3 and ERK signalling on invasive ability of gastric cancer cells were examined using a small-interfering RNA system and specific inhibitors. AGS cells were co-cultured with cancer-associated fibroblasts (CAFs) from human gastric cancer tissues and assessed by invasion assay. RESULTS: Interleukin-22 and its receptor were expressed in α-smooth muscle actin-positive stromal cells and tumour cells at the invasive front of gastric cancer tissues, respectively. The expression of IL-22 and IL-22R1 was significantly related to lymphatic invasion. Interleukin-22 treatment promoted the invasive ability of gastric cancer cells through STAT3 and ERK activation. The invasive ability of gastric cancer cells was significantly enhanced by co-culture with IL-22-expressing CAFs. CONCLUSIONS: Interleukin-22 produced by CAFs promotes gastric cancer cell invasion via STAT3 and ERK signalling.

Functional analysis of platelet-derived growth factor receptor-ß in neural stem/progenitor cells.

Activation of neural stem/progenitor cells (NSPCs) is a potential therapeutic strategy of neurological disorders. In this study, NSPCs of subventricular zone were isolated and cultured from platelet-derived growth factor-ß-receptor-knockout (PDGFR-ß(-/-)) mice of postnatal day 1 (P1) and P28, and the roles of PDGFR-ß were examined in these cells. In PDGFR-ß-preserving control NSPCs, stem cell activities, such as numbers and diameters of secondary neurospheres, cell proliferation and survival rates, were significantly higher in P1 NSPCs than those in P28 NSPCs. In PDGFR-ß(-/-) NSPCs, most of these parameters were decreased as compared with age-matched controls. Among them, the decrease of secondary neurosphere formation was most striking in P1 and P28 PDGFR-ß(-/-) NSPCs and in P28 control NSPCs as compared with P1 control NSPCs. PCR-array and following quantitative real-time PCR (qRT-PCR) analyses demonstrated that expressions of fibroblast growth factor-2 (FGF2) and exons IV-IX of brain-derived neurotrophic factor (BDNF) were decreased, and noggin was increased in P1 PDGFR-ß(-/-) as compared with P1 controls. Addition of BDNF rescued the number and diameter of secondary neurospheres in P1 PDGFR-ß(-/-) NSPCs to similar levels as controls. The expressions of PDGFs and PDGFRs in control NSPCs were increased along with the differentiation-induction, where phosphorylated PDGFR-ß was co-localized with neuronal and astrocyte differentiation markers. In controls, the neuronal differentiation was decreased, and the glial differentiation was increased from P1 to P28 NSPCs. Compared with P1 controls, neuronal differentiation was reduced in P1 PDGFR-ß(-/-) NSPCs, whereas glial differentiation was comparable between the two genotypes. These results suggest that PDGFR-ß signaling is important for the self-renewal and multipotency of NSPCs, particularly in neonatal NSPCs. BDNF, FGF2, and noggin may be involved in the effects of PDGFR-ß signaling in these cells. Accordingly, the activation of PDGFR-ß in NSPCs may be a novel therapeutic strategy of neurological diseases.

Formation of chlorinated aromatics in model fly ashes using various copper compounds.

Various copper compounds found in fly ash are related to the formation of chlorinated aromatics. The formation potentials of chlorinated aromatics in different model fly ashes containing various copper compounds and the chemical behavior of such copper compounds were investigated. In model fly ash with copper metal, hydroxide, carbonate, or oxides, the generated amounts of chlorobenzene (CBz) and polychlorinated biphenyls (PCBs) and the average chlorination numbers were low and at the same level, respectively. The maximum generated amounts of chlorinated aromatics were observed at 300 degrees C. Although X-ray absorption near edge structure (XANES) spectra indicated that the chemical form of copper compounds changed little, they were found to promote the formation of chlorinated aromatics. Therefore, these copper compounds play the same role as CuO. On the other hand, in model fly ash with copper chloride, the generated amounts of CBz and PCBs were quite high and the average chlorination numbers was high. Dynamic changes were observed in XANES spectra, and the pre-edge peak attributed to monovalent copper compounds appeared at around 300 degrees C. A large difference was observed between these two groups in the amount of CuCl generated and the homologs of chlorinated aromatics present, indicating that CuCl played an important role in the formation of chlorinated aromatics.

[Three cases of spontaneous mediastinal emphysema].

We report 3 cases of spontaneous mediastinal emphysema. All patients were young males, and had predisposing episodes for development of spontaneous mediastinal emphysema; sports in 2, loud voice in 1. The each chief complaint was dyspnea, throat pain, and epigastric pain. Two patients were admitted, but 1 rejected admission despite sufficient informed consent. All patients became asymptomatic with mediastinal air reabsorption within a week. We should recognize spontaneous mediastinal emphysema as one cause of chest, back, neck and epigastric pain.

Role of Necl-5 in the pathophysiology of colorectal lesions induced by dimethylhydrazine and/or dextran sodium sulphate.

Necl-5 is an immunoglobulin-like molecule that was originally identified as a poliovirus receptor. Although Necl-5 expression is often up-regulated in cancer cells, its pathophysiological significance in the development of cancer remains unclear. We investigated the roles of Necl-5 in the development of colitis-associated neoplasia. Necl-5-deficient mice were generated and treated with dimethylhydrazine (DMH) and/or dextran sodium sulphate (DSS) to induce colitis and its associated neoplasias. Colon tissues were examined for histology, Ki-67 expression by immunohistochemistry and K-ras gene mutation. Colon tumours occurred significantly less frequently in heterozygous (Necl-5(+/-)) or homozygous Necl-5-deficient (Necl-5(-/-)) mice than in wild-type (WT) mice with DMH/DSS treatment. Total ulcer index and inflammatory cell infiltration were significantly lower in Necl-5(-/-) mice than in WT mice with DSS alone or DMH/DSS treatment. Colon tumours in both WT and Necl-5(-/-) mice showed high cell proliferation ability but lacked K-ras mutation. The total Ki-67 labelling index in non-neoplastic colon epithelium was significantly higher in WT (45.9 +/- 0.94) than in Necl-5(+/-) (34.3 +/- 1.40) or Necl-5(-/-) (27.7 +/- 1.15) mice with DMH/DSS treatment (p < 0.001). Necl-5 plays a role in the development of colitis-associated cancer by up-regulating colonic mucosal cell proliferation.

Involvement of REG Ialpha protein in the regeneration of ductal epithelial cells in the minor salivary glands of patients with Sjögren's syndrome.

The regenerating gene (Reg) was originally isolated from regenerating rat pancreatic islets and revealed recently to constitute a multi-gene family in humans. REG Ialpha protein is known to be overexpressed not only in various human inflammatory diseases but also in various experimental models of inflammation in animal tissues. However, its involvement in pathophysiology of the minor salivary gland (MSG) is not clear. We investigated REG Ialpha expression in the MSG of patients with primary Sjögren's syndrome (SS) and assessed its role in ductal epithelial cell proliferation in such tissues. Lip biopsy specimens were obtained from 40 patients with primary SS and examined using immunohistochemistry for REG Ialpha protein, Ki67 and single-strand DNA (ssDNA). The relationships among clinicopathological factors and expression of REG Ialpha protein, Ki67 and ssDNA in the MSG were then analysed. REG Ialpha protein was expressed rarely in ductal epithelial cells of the normal MSG but was apparently overexpressed in those of patients with SS. The labelling indices for both Ki67 and ssDNA in the ductal cells of the MSGs were significantly higher in SS patients than in controls. Moreover, these labelling indices were significantly higher in REG Ialpha-positive than in negative SS patients. REG Ialpha protein may play a role in the regeneration of ductal epithelial cells in the MSGs of patients with SS.

EXTL3 promoter methylation down-regulates EXTL3 and heparan sulphate expression in mucinous colorectal cancers.

Exostoses like-3 (EXTL3) is a putative tumour suppressor gene but its involvement in colorectal cancer (CRC) is unclear. We have investigated the role of methylation of the EXTL3 promoter as a mechanism for EXTL3 regulation and tested the hypothesis that loss of EXTL3 expression is associated with mucinous differentiation and alteration of glycoprotein expression in CRC cells. The methylation status of the EXTL3 gene promoter was analysed by methylation-specific PCR following bisulphite modification in CRC cell lines and microdissected primary CRC tissues and their corresponding adjacent normal colorectal mucosa. EXTL3 promoter methylation was detected in seven of 11 mucinous CRCs (63.6%) but in none of 26 non-mucinous CRCs examined. EXTL3 promoter methylation was also detected in the normal colonic mucosa of three patients with mucinous CRC but not in the normal colonic mucosa of any patients with non-mucinous CRC. The presence of EXTL3 methylation was significantly associated with the partial loss of HS expression in mucinous CRC lesions. The mucinous CRC cell lines, Colo201 and Colo205, showed EXTL3 promoter methylation and loss of EXTL3 mRNA expression. However 5-aza-2'-deoxycytidine treatment demethylated the EXTL3 gene promoter and restored its mRNA expression. Furthermore, the basal expression of HS in CRC cells was abolished by treatment with EXTL3-siRNA. We conclude that EXTL3 promoter methylation and its related loss of EXTL3 expression are involved in the loss of HS expression in mucinous CRCs.

Expression of SDF-1 alpha and nuclear CXCR4 predicts lymph node metastasis in colorectal cancer.

Although stromal cell-derived factor (SDF)-1 alpha and its receptor CXCR4 are experimentally suggested to be involved in tumorigenicity, the clinicopathological significance of their expression in human disease is not fully understood. We examined SDF-1 alpha and CXCR4 expression in colorectal cancers (CRCs) and their related lymph nodes (LNs), and investigated its relationship to clinicopathological features. Specimens of 60 primary CRCs and 27 related LNs were examined immunohistochemically for not only positivity but also immunostaining patterns for SDF-1 alpha and CXCR4. The relationships between clinicopathological features and SDF-1 alpha or CXCR4 expression were then analysed. Stromal cell-derived factor-1 alpha and CXCR4 expression were significantly associated with LN metastasis, tumour stage, and survival of CRC patients. Twenty-nine of 47 CXCR4-positive CRCs (61.7%) showed clear CXCR4 immunoreactivity in the nucleus and a weak signal in the cytoplasm (nuclear type), whereas others showed no nuclear immunoreactivity but a diffuse signal in the cytoplasm and at the plasma membrane (cytomembrane type). Colorectal cancer patients with nuclear CXCR4 expression showed significantly more frequent LN metastasis than did those with cytomembrane expression. Colorectal cancer patients with nuclear CXCR4 expression in the primary lesion frequently had cytomembrane CXCR4-positive tumours in their LNs. In conclusion, expression of SDF-1 alpha and nuclear CXCR4 predicts LN metastasis in CRCs.

Determining the treatment strategy for colorectal neoplastic lesions: endoscopic assessment or the non-lifting sign for diagnosing invasion depth?

BACKGROUND AND STUDY AIMS: Assessment of the invasion depth of colorectal neoplasia is important in deciding between endoscopic and surgical resection treatment methods. Prior to attempting endoscopic resection, the lesion is lifted by submucosal injection, and a positive "non-lifting sign" is usually considered to indicate deeper submucosal infiltration. The purpose of this prospective multicenter study was to assess the predictive value of the non-lifting sign for differentiating between adenoma and early cancer (up to discrete submucosal infiltration [sm1]) and cancer with deeper infiltration (sm2). PATIENTS AND METHODS: During an 11-month period, a total of 271 colorectal neoplastic lesions in 239 patients were included in the study. Apart from the location, size, and macroscopic type of the lesion, the presence or absence of the non-lifting sign was recorded and compared with the endoscopic assessment of invasion depth. RESULTS: The non-lifting sign had a sensitivity of 61.5 %, a specificity of 98.4 %, a positive predictive value of 80.0 %, a negative predictive value of 96.0 %, and an accuracy of 94.8 %. Endoscopic diagnosis of deeper infiltration had a sensitivity of 84.6 %, a specificity of 98.8 %, a positive predictive value of 88.0 %, a negative predictive value of 98.4 %, and an accuracy of 97.4 %. Statistically significant differences were found in terms of sensitivity and accuracy. CONCLUSION: Because of its lower sensitivity and accuracy, the non-lifting sign will not replace endoscopic assessment. If a lesion does not lift, this can make resection technically difficult, but does not reliably predict deeper cancerous invasion.

Acetylsalicylic acid enhances antiproliferative effects of the EGFR inhibitor gefitinib in the absence of activating mutations in gastric cancer.

The epidermal growth factor receptor (EGFR) is highly expressed in gastric cancer indicating its suitability as a target for receptor tyrosine kinase (RTK) inhibitors. In the current study we explored the role of EGFR and its potential use as a therapeutic target in gastric cancer. First we analyzed 66 gastric cancer samples of Asian and Caucasian patients for the presence of EGFR mutations. No activating EGFR mutations were found and gefitinib alone was only weakly effective in gastric cancer cell lines. However, acetylsalicylic acid (ASA) significantly enhanced the inhibitory effects of gefitinib indicating synergistic action. Whole genome expression profiling indicated significant regulation of 120 genes in the case of co-administration of gefitinib and ASA (32 induced, 88 repressed) in gastric adenocarcinoma cells. Further analyses indicated that several important signalling pathways were effectively inhibited by simultaneous exposure to gefitinib and ASA. Our findings indicate that although gastric cancer does not seem to harbour mutations which render the cancer cells constitutively susceptible to gefitinib, the co-administration of ASA can strengthen RTK inhibitor activity in adenocarcinoma cells by EGFR activation. This is the first report of effective modulation of EGFR-inhibition activity in cancer.