A single intra-articular dose of vitamin D analog calcipotriol alleviates synovitis without adverse effects in rats.

1,25-dihydroxyvitamin-D3 and its derivatives have shown anti-arthritic and chondroprotective effects in experimental animal models with prophylactic dosing. The purpose of this preliminary study was to test the efficacy and safety of calcipotriol, vitamin D analog, as a treatment for a fully-developed knee arthritis in Zymosan-induced arthritis (ZIA) model. Forty 5-month-old male Sprague-Dawley rats were randomized into three arthritis groups and a non-arthritic control group with no injections (10 rats/group). A day after Zymosan (0.1 mg) had been administrated into the right knee joints, the same knees were injected with calcipotriol (0.1 mg/kg), dexamethasone (0.1 mg/kg) or vehicle in a 100 µl volume. The left control knees were injected with saline (PBS) on two consecutive days. All injections, blood sampling and measurements were performed under general anesthesia on days 0, 1, 3 and 8. Internal organs and knees were harvested on day 8 and the histology of the whole knees was assessed blinded. Joints treated with calcipotriol showed a milder histological synovitis than those treated with vehicle (p = 0.041), but there was no statistically significant difference between the dexamethasone and vehicle groups. The clinical severity of arthritis did not differ between the arthritis groups measured by body temperature, swelling of the knee, thermal imaging, clinical scoring or cytokine levels on days 1, 3 and 8. Weight loss was bigger in rats treated with dexamethasone, propably due to loss of appetite,compared to other arthritis groups on days 2-3 (p<0.05). Study drugs did not influence serum calcium ion and glucose levels. Taken together, this preliminary study shows that a single intra-articular injection of calcipotriol reduces histological grade of synovitis a week after the local injection, but dexamethasone did not differ from the vehicle. Calcipotriol may have an early disease-modifying effect in the rat ZIA model without obvious side effects.

Tenascin C, Fibronectin, and Tumor-Stroma Ratio in Pancreatic Ductal Adenocarcinoma.

OBJECTIVES: Pancreatic ductal adenocarcinoma (PDAC) is characterized by abundant stroma with increased expression of tenascin C and fibronectin. Their role and tumor-stroma ratio in PDAC are not well known. The aim of this study was to evaluate tenascin C and fibronectin expression and tumor-stroma ratio and their prognostic relevance in PDAC. METHODS: Ninety-five resected PDACs were immunohistochemically stained for tenascin C and fibronectin, and the expression was separately assessed in tumor bulk and front. Tumor-stroma ratio was determined with sections stained with hematoxylin-eosin. RESULTS: Tenascin C and fibronectin were abundantly expressed in the stroma of PDAC, but absent in adjacent normal pancreatic tissue. Fibronectin expression of the bulk was associated with high T class (P = 0.045). In the main analysis, tenascin C and fibronectin expression and tumor-stroma ratio were not associated with patient survival. In a subgroup analysis of early-stage PDAC (T1-T2 tumors), high tenascin C expression in the tumor bulk was associated with poor prognosis (hazard ratio, 8.23; 95% confidence interval, 2.71-24.96). CONCLUSIONS: Tenascin C and fibronectin are abundantly expressed in PDAC, but they seem to have no major association with patient survival. However, in early-stage PDAC, tenascin C expression of the tumor bulk may have prognostic impact. Tumor-stroma ratio has no prognostic value in PDAC.

Toll-like receptors 2, 4 and 9 and hypoxia markers HIF-1alpha and CAIX in pancreatic intraepithelial neoplasia.

Pancreatic cancer arises from precursor lesions called pancreatic intraepithelial neoplasia (PanIN) characterized by inflammatory microenvironment. In pancreatic cancer, strong innate immunity and hypoxia responses are typical. Occurrence and relationship of these responses in human PanINs is unknown. We have studied the expression of toll-like receptors (TLR) TLR2, TLR4 and TLR9, and hypoxia markers HIF-1alpha and Carbonic anhydrase IX (CAIX) in normal and inflamed pancreatic ducts, in PanINs and in cancers. The samples of 69 surgically resected pancreatic ductal adenocarcinoma patients were stained using immunohistochemistry. We found TLR2, TLR9, HIF-1alpha and CAIX to be prominently expressed in pancreatic intraepithelial neoplasia. Expression of TLR2 showed a linear increase from PanIN1 to PanIN3, while the highest TLR4 expression was detected in inflamed ducts, and TLR9 expression in PanIN1 lesions. Within the PanIN1-group, nuclear HIF-1alpha correlated with membranous and cytoplasmic TLR2 expression (ρ = 0.982 and 0.815; p < 0.001 and p = 0.025, respectively), and in the PanIN2-group nuclear HIF-1alpha correlated with nuclear TLR9 expression 0.636, p = 0.026). Our findings show that the expression of TLRs 2, 4 and 9, and hypoxia markers HIF-1alpha and CAIX is abnormal in pancreatic intraepithelial neoplasia suggesting that both the innate immunity activation and hypoxia response are involved in early pancreatic carcinogenesis. However, these processes might be independent.

Weak HIF-1alpha expression indicates poor prognosis in resectable pancreatic ductal adenocarcinoma.

BACKGROUND: HIF-1alpha and CAIX proteins are commonly expressed under hypoxic conditions, but other regulatory factors have been described as well. Pancreatic ductal adenocarcinoma (PDAC) is characterized by hypoxia and strong stromal reaction and has a dismal prognosis with the currently available treatment modalities. METHODS: We investigated the expression and prognostic role of HIF-1alpha and CAIX in PDAC series from Northern Finland (n = 69) using immunohistochemistry. RESULTS: In our PDAC cases, 95 and 85% showed HIF-1alpha and CAIX expression, respectively. Low HIF-1alpha expression correlated with poor prognosis, and multivariate analysis identified weak HIF-1alpha intensity as an independent prognostic factor for PDAC-specific deaths (HR 2.176, 95% CI 1.216-3.893; p = 0.009). There was no correlation between HIF-1alpha and CAIX expression levels, and the latter did not relate with survival. CONCLUSIONS: Our findings are in contrast with previous research by finding an association between low HIF-1alpha and poor prognosis. The biological mechanisms remain speculative, but such an unexpected relation with prognosis and absence of correlation between HIF-1alpha and CAIX suggests that the prognostic association of HIF-1alpha may not directly be linked with hypoxia. Accordingly, the role of HIF-1alpha might be more complex than previously thought and the use of this marker as a hypoxia-related prognostic factor should be addressed with caution.

Osteogenic Differentiation of Human Mesenchymal Stem cells in a 3D Woven Scaffold.

Fiber-based scaffolds produced by textile manufacturing technology offer versatile materials for tissue engineering applications since a wide range of crucial scaffold parameters, including porosity, pore size and interconnectivity, can be accurately controlled using 3D weaving. In this study, we developed a weavable, bioactive biodegradable composite fiber from poly (lactic acid) (PLA) and hydroxyapatite powder by melt spinning. Subsequently, scaffolds of these fibers were fabricated by 3D weaving. The differentiation of human mesenchymal stem cells (hMSCs) in vitro was studied on the 3D scaffolds and compared with differentiation on 2D substrates having the same material composition. Our data showed that the 3D woven scaffolds have a major impact on hMSCs proliferation and activation. The 3D architecture supports the differentiation of the hMSCs into osteoblast cells and enhances the production of mineralized bone matrix. The present study further confirms that a 3D scaffold promotes hMSCs differentiation into the osteoblast-lineage and bone mineralization.

Tenascin-C and fibronectin in normal esophageal mucosa, Barrett's esophagus, dysplasia and adenocarcinoma.

BACKGROUND: Tenascin-C and fibronectin are adhesive glycoproteins modulating the structure of the extracellular matrix and cellular functions. Their expression and function in esophageal adenocarcinoma is poorly known. The aim of this study was to evaluate the expression of tenascin-C and fibronectin in esophageal adenocarcinoma and its precursor stages. RESULTS: Stromal tenascin-C and fibronectin expression were found in all evaluated lesion types. Expression of both molecules increased from gastric metaplasia towards adenocarcinoma (p<0.05). In carcinomas, tenascin-C expression in the bulk was associated with T-stage (p=0.006), presence of lymph node (p=0.004) and distant organ metastases (p=0.007). Abundant tenascin-C expression associated with poor survival (p=0.034) in univariate analysis. Fibronectin expression associated to T-stage (p=0.030). Expression of tenascin-C or fibronectin in the tumor invasive front was not associated to clinicopathological variables or survival. No significant correlation with tumor/stroma percentage, cancer-associated fibroblasts or mean vascular density was observed with either tenascin-C or fibronectin. METHODS: Tenascin-C and fibronectin were stained immunohistochemically and assessed in esophageal specimens from patients with esophageal adenocarcinoma (n=90) or dysplasia (n=30). Structures and lesion were evaluated including normal esophagus (n=77), gastric (n=61) or intestinal (n=51) metaplasia without dysplasia, and low-grade (n=42) or high-grade (n=34) dysplasia, and esophageal adenocarcinoma (n=90). In carcinomas, both bulk and invasive front were separately evaluated. In addition, tumor/stroma percentage, cancer-associated fibroblasts and mean vascular density were evaluated. CONCLUSIONS: Tenascin-C and fibronectin are upregulated in esophageal adenocarcinoma when compared to Barrett's esophagus and dysplasia. Increased tenascin-C expression is associated with metastasis and poor prognosis in esophageal adenocarcinoma.

Intratumoral lactate metabolism in Barrett's esophagus and adenocarcinoma.

BACKGROUND: Monocarboxylate transporters (MCTs) are cell membrane proteins which transport pyruvate, lactate and ketone bodies across the plasma membrane. MCTs are activated in various cancers, but their expression in esophageal adenocarcinoma is not known. The present study was conducted to elucidate the expression of MCTs in esophageal adenocarcinoma and its precursor lesions. RESULTS: Cytoplasmic MCT1, MCT4 and MTCO1 expression linearly increased from normal epithelium to Barrett's mucosa to dysplasia and cancer. Low cytoplasmic MCT1 expression associated with high T-class (P < 0.01), positive lymph node metastases (P < 0.05), positive distant metastases (P < 0.01) and high tumor stage (P < 0.01). High cytoplasmic MCT4 expression correlated significantly with positive distant metastases (P < 0.05). Both low MCT1 and high MCT4 histoscore predicted survival in univariate analysis (P < 0.01). MCT4 histoscore predicted survival in multivariate analysis (P = 0.043; HR 1.8 95%CI 1.0-3.1). MTCO1 expression was not correlated to clinicopathological variables or survival. MATERIALS AND METHODS: MCT1, MCT4 and mitochondrial cytochrome c oxidase (MTCO1) expression were determined with immunohistochemistry in esophageal specimens from 129 patients with columnar dysplasia or adenocarcinoma. Specimens including normal esophagus (n = 88), gastric (n = 67) or intestinal metaplasia (n = 51), low-grade (n = 42), high-grade dysplasia (n = 37) and esophageal adenocarcinoma (n = 99) were evaluated. CONCLUSIONS: Major increase in markers of tumor metabolism occurs during carcinogenesis and progression of esophageal adenocarcinoma. MCT1 and MCT4 are prognostic factors in esophageal adenocarcinoma.

Localization of nucleic acid-sensing toll-like receptors in human and mouse pancreas.

Nucleic acid-sensing toll-like receptors (TLRs) (3, 7, 8, 9) have a role both in antiviral innate immunity and in autoimmune disorders. We assessed the expression of TLR3, 7, 8 and 9 in human and mouse pancreas focusing on the subpopulations of cells in the Langerhans islets. We studied eight human samples with normal pancreatic islets and two samples from patients with type 1 diabetes. Additionally, 10 CD-1 mouse pancreases were analysed. Immunohistochemical double-stainings for the TLRs and insulin, glucagon or somatostatin, respectively, were performed along with appropriate controls. In human pancreas, strong immunoreaction of TLR7 and TLR8 was observed in the insulin-positive beta cells, whereas glucagon- or somatostatin-expressing cells of the islets were weakly stained or negative. In type 1 diabetes, the expression in islets was weak or lost (TLR7: p = 0.014, TLR8: p = 0.053), correlating with loss of beta cells. TLR3 and 9 were expressed only weakly with no correlation with specific cell types. In mouse pancreas, only TLR9 was detected. Intra-pancreatic nerve ganglia strongly expressed TLR7. The strong expression of TLR7 and TLR8 in the beta cells of normal human islets could be an important piece in the puzzle of type 1 diabetes pathogenesis, and be linked with destruction of this particular subpopulation of the islet cells. In normal mice, only TLR9 can be constantly detected in the islets, highlighting differences between the species.

Doublecortin-like kinase 1-positive enterocyte - a new cell type in human intestine.

Doublecortin-like kinase 1 (DCLK1) is a microtubule-associated kinase. In murine intestine, DCLK1 marks tuft cells with characteristic microvilli, features of neuroendocrine cells and also quiescent stem cell-like properties. The occurrence and pathological role of DCLK1-positive cells in human intestinal mucosa is unknown. We analysed DCLK1 expression in healthy duodenal, jejunal and colorectal mucosa samples (n = 35), and in duodenal specimens from patients with coeliac disease (n = 20). The samples were immunohistochemically double-stained with DCLK1, and synaptophysin, chromogranin A and Ki-67. Ultrastructure of DCLK1-expressing duodenal cells was assessed using correlative light and electron microscopy. DCLK1 expression was seen in about 1% of epithelial cells diffusely scattered through the intestinal epithelium. Electron microscopy showed that the duodenal DCLK1-positive cells had short apical microvilli similar to neighbouring enterocytes and cytoplasmic granules on the basal side. DCLK1-positive cells were stained with synaptophysin. The number of DCLK1-positive cells was decreased in villus atrophy in coeliac disease. Our findings indicate that in human intestinal epithelium, DLCK1-positive cells form a subpopulation of non-proliferating neuroendocrine cells with apical brush border similar to that in enterocytes, and their number is decreased in untreated coeliac disease.

Nucleic acid-sensing toll-like receptors 3, 7 and 8 in esophageal epithelium, barrett's esophagus, dysplasia and adenocarcinoma.

Toll-like receptors (TLRs) are immunological receptors recognizing various microbial and endogenous ligands, such as DNA, RNA, and other microbial and host components thus activating immunological responses. The expression of TLRs in esophageal adenocarcinoma is not well known. The aim of this study was to evaluate expression patterns of those TLRs that sense nucleic acids in Barrett's esophagus with and without dysplasia and in esophageal adenocarcinoma. TLRs 3, 7 and 8 were stained immunohistochemically and evaluated in a cohort of patients with esophageal adenocarcinoma or dysplasia. Specimens with normal esophagus (n = 88), gastric (n = 67) or intestinal metaplasia (n = 51) without dysplasia, and low-grade (n = 42) or high-grade dysplasia (n = 37) and esophageal adenocarcinoma (n = 99) were studied. We used immunofluorescence to confirm the subcellular localization of TLRs. We found abundant expression of TLR3, 7 and 8 in esophageal squamous epithelium, columnar metaplasia, dysplasia and adenocarcinoma. Cytoplasmic expression of TLR3, TLR7 or TLR8 did not associate to clinicopathological parameters or prognosis in esophageal cancer. High nuclear expression of TLR8, confirmed with immunofluorescence, in cancer cells was observed in tumors of high T-stage (p < 0.01) and in tumors with organ metastasis (p < 0.001). High nuclear TLR8 expression was associated with poor prognosis (p < 0.001). The expression of TLR3, TLR7 and TLR8 increased toward dysplasia and adenocarcinoma. We demonstrated nuclear localization of TLR8, which associates with metastasis and poor prognosis. TLR3 and TLR7 do not seem to have prognostic significance in esophageal adenocarcinoma.

Nuclear localization of Toll-like receptor 5 in Barrett's esophagus and esophageal adenocarcinoma is associated with metastatic behavior.

Toll-like receptor 5 (TLR5) recognizes bacterial flagellin. Increased cytoplasmic and nuclear expression of some TLRs has been previously reported in dysplasia in Barrett's esophagus and various malignant lesions in association with survival and metastasis. We assessed nuclear expression of TLR5 in Barrett's esophagus and esophageal adenocarcinoma (n = 94) by immunohistochemistry and immunofluorescent staining. Nuclear expression was observed in the majority of studied lesions with high variation in the proportion of positive nuclei. Expression in lymph node metastases was significantly higher than in esophageal lesions (p < 0.05), except for intestinal metaplasia and low-grade dysplasia. Furthermore, nuclear expression of TLR5 was associated with the presence of lymph node metastases (p = 0.033). In conclusion, we report nuclear TLR5 expression in Barrett's esophagus and adenocarcinoma. Abundance of positive nuclei in association with lymph node metastases suggests that TLR5 is involved in the pathogenesis and dissemination of esophageal adenocarcinoma through as-yet-uncharacterized mechanisms.

The Expression of Toll-like Receptors in Normal Human and Murine Gastrointestinal Organs and the Effect of Microbiome and Cancer.

Toll-like receptors (TLRs) are innate immune receptors expressed in all parts of the alimentary tract. However, analyses comparing expression in different segments and data on germ-free animals are lacking. Alimentary tract cancers show increased TLR expression. According to the field effect concept, carcinogenetic factors induce subtle cancer predisposing alterations in the whole organ. We studied TLR1 to TLR9 expression in all segments of the alimentary tract from cancer patients' tumor-adjacent normal mucosa, healthy organ donors, and conventional and germ-free mice by using immunohistochemistry and quantitative PCR. All TLRs were expressed in all segments of the alimentary tract. Expression was most intensive in the small intestine in humans and conventional mice, but germ-free mice showed less expression in the small intestine. TLR expression levels were similar in cancer patients and organ donors. We provide systematic baseline data on the TLR expression in the alimentary tract. Normal epithelium adjacent to tumor seems to have similar TLR expression compared with healthy tissues suggesting absence of any field effect in TLR expression. Accordingly, specimens from cancer patients' normal tumor-adjacent tissue can be used as control tissues in immunohistochemical TLR studies in gastrointestinal cancer.

Toll-like receptors 1, 2, 4 and 6 in esophageal epithelium, Barrett's esophagus, dysplasia and adenocarcinoma.

BACKGROUND: Toll-like receptors (TLRs) recognize microbial and endogenous ligands and have already shown to play a role in esophageal cancer. In this study, we evaluated especially TLRs that sense bacterial cell wall components in Barrett's esophagus, dysplasia and esophageal adenocarcinoma. METHODS: TLRs 1, 2, 4 and 6 were stained immunohistochemically and assessed in esophageal specimens from patients with esophageal dysplasia (n = 30) or adenocarcinoma (n = 99). Structures and lesions were evaluated including normal esophagus (n = 88), gastric (n = 67) or intestinal metaplasia (n = 51) without dysplasia, and low-grade (n = 42) or high-grade dysplasia (n = 37), and esophageal adenocarcinoma (n = 99). RESULTS: We found TLR1, TLR2, TLR4 and TLR6 expression in all lesions. TLR expression increased in Barrett's mucosa and dysplasia. There was profound increase of TLR expression from gastric- to intestinal-type columnar epithelium. In cancers, high nuclear and cytoplasmic staining of TLR4 associated with metastatic disease and poor prognosis. CONCLUSIONS: TLR1, TLR2, TLR4 and TLR6 are upregulated during malignant changes of esophageal columnar epithelium. Increased TLR4 expression associates with advanced stage and poor prognosis in esophageal adenocarcinoma.

Telomeric G-quadruplex-forming DNA fragments induce TLR9-mediated and LL-37-regulated invasion in breast cancer cells in vitro.

Toll-like receptor 9 (TLR9) is a cellular DNA-receptor widely expressed in cancers. We previously showed that synthetic and self-derived DNA fragments induce TLR9-mediated breast cancer cell invasion in vitro. We investigated here the invasive effects of two nuclease-resistant DNA fragments, a 9-mer hairpin, and a G-quadruplex DNA based on the human telomere sequence, both having native phosphodiester backbone. Cellular uptake of DNAs was investigated with immunofluorescence, invasion was studied with Matrigel-assays, and mRNA and protein expression were studied with qPCR and Western blotting and protease activity with zymograms. TLR9 expression was suppressed through siRNA. Although both DNAs induced TLR9-mediated changes in pro-invasive mRNA expression, only the telomeric G-quadruplex DNA significantly increased cellular invasion. This was inhibited with GM6001 and aprotinin, suggesting MMP- and serine protease mediation. Furthermore, complexing with LL-37, a cathelicidin-peptide present in breast cancers, increased 9-mer hairpin and G-quadruplex DNA uptake into the cancer cells. However, DNA/LL-37 complexes decreased invasion, as compared with DNA-treatment alone. Invasion studies were conducted also with DNA fragments isolated from neoadjuvant chemotherapy-treated breast tumors. Also such DNA induced breast cancer cell invasion in vitro. As with the synthetic DNAs, this invasive effect was reduced by complexing the neoadjuvant tumor-derived DNAs with LL-37. We conclude that 9-mer hairpin and G-quadruplex DNA fragments are nuclease-resistant DNA structures that can act as invasion-inducing TLR9 ligands. Their cellular uptake and the invasive effects are regulated via LL-37. Although such structures may be present in chemotherapy-treated tumors, the clinical significance of this finding requires further studying.

Toll-like receptor 9 mediates invasion and predicts prognosis in squamous cell carcinoma of the mobile tongue.

BACKGROUND: Toll-like receptor 9 (TLR9) is a cellular receptor, which recognizes bacterial and host-derived DNA. Stimulation of TLR9 induces cellular invasion via matrix metalloproteinase 13 (MMP-13). The aim of this study was to evaluate the role of TLR9 in invasion of oral tongue squamous cell carcinoma (OTSCC). METHODS: The effects of TLR9 ligands on oral squamous cell carcinoma cell lines were studied with invasion and migration assays, as well as in a myoma organotypic model. RESULTS: The TLR9 ligand, CpG-ODN, increased invasion and migration in OTSCC lines. These effects were reduced by TLR9 siRNA or inhibition with TLR9 antibodies. Immunohistochemical analysis of tissues from 195 patients with OTSCC revealed that TLR9 was expressed in 181/195 carcinomas. The expression of TLR9 was higher in the malignant cells than in the normal epithelium. High TLR9 expression was associated with high MMP-13 expression and poor differentiation. High TLR9 expression was also identified as an independent predictor of poor prognosis (HR 1.810, 95% CI [1.053-3.112]). CONCLUSION: Toll-like receptor 9 mediates OTSCC invasion and migration in vitro and is an independent prognostic factor of OTSCC. Inhibition of TLR9 may be a novel therapeutic opportunity in oral cancer.

Effect of bioactive extruded PLA/HA composite films on focal adhesion formation of preosteoblastic cells.

The quality of the initial cell attachment to a biomaterial will influence any further cell function, including spreading, proliferation, differentiation and viability. Cell attachment is influenced by the material's ability to adsorb proteins, which is related to the surface chemistry and topography of the material. In this study, we incorporated hydroxyapatite (HA) particles into a poly(lactic acid) (PLA) composite and evaluated the surface structure and the effects of HA density on the initial cell attachment in vitro of murine calvarial preosteoblasts (MC3T3-EI). Scanning electron microscopy (SEM), atomic force microscopy (AFM) and infrared spectroscopy (FTIR) showed that the HA particles were successfully incorporated into the PLA matrix and located at the surface which is of importance in order to maintain the bioactive effect of the HA particles. SEM and AFM investigation revealed that the HA density (particles/area) as well as surface roughness increased with HA loading concentration (i.e. 5, 10, 15 and 20wt%), which promoted protein adsorption. Furthermore, the presence of HA on the surface enhanced cell spreading, increased the formation of actin stress fibers and significantly improved the expression of vinculin in MC3T3-E1 cells which is a key player in the regulation of cell adhesion. These results suggest the potential utility of PLA/HA composites as biomaterials for use as a bone substitute material and in tissue engineering applications.

Increased Toll-like receptor 5 expression indicates esophageal columnar dysplasia.

Toll-like receptor 5 (TLR5) is an immune receptor, which recognizes bacterial flagellin. Increased expression has been reported in various premalignant and malignant lesions indicating a role in carcinogenesis. We assessed the expression of TLR5 in normal esophageal squamous epithelium, Barrett's esophagus with and without dysplasia, and in esophageal adenocarcinoma. Specimens with normal esophagus (n=93), gastric (n=75) or intestinal metaplasia (n=53) without dysplasia, and low-grade (n=56) or high-grade dysplasia (n=33) and esophageal adenocarcinoma (n=94) were studied. TLR5 immunohistochemical stainings were analyzed for the proportion of positive cells and the intensity of expression. In normal squamous epithelium, only the basal third showed TLR5 expression. In esophageal gastric or intestinal metaplasia, expression was present in majority of the cells but significantly weaker (p<0.001) than in dysplastic epithelium. In dysplasia, expression extended to the apical cytoplasm, contrasting basolateral expression in non-dysplastic columnar epithelium. Receiver operating characteristic curve analysis showed that moderate to high expression intensity of TLR5 indicates low-grade dysplasia with 86 % sensitivity and 83 % specificity. Carcinomas showed increased expression in comparison with non-dysplastic columnar epithelium, but there was no association with prognosis. Our results indicate that the esophageal columnar dysplasia is associated with clear increase of TLR5 expression and dissolution of regular polarized expression. TLR5 staining provides a possible biomarker for the recognition of low-grade dysplasia. In addition, the findings suggest a role for abnormal expression of TLR5 in the pathogenesis of esophageal adenocarcinoma and suggest importance of altered microbiome in the pathogenesis of complications of Barrett's esophagus.

Short DNA sequences and bacterial DNA induce esophageal, gastric, and colorectal cancer cell invasion.

Toll-like receptor 9 (TLR9) recognizes both bacterial and self-DNA and it is abundantly expressed in the gastrointestinal tract. In this study, we investigated the influences of both bacterial DNA and specific short DNA sequences on TLR9-mediated gastrointestinal cancer cell invasion. We assessed the effect of various DNA ligands on cellular invasion and on TLR9 and matrix metalloproteinase expression of three gastrointestinal cancer cell lines. DNA-ligands described in this study include CpG-ODN M362, 9-mer (hairpin), human telomeric sequence h-Tel22 G-quadruplex, and bacterial DNAs from Escherichia coli and Helicobacter pylori. All of the DNAs studied were demonstrated to induce invasion in the studied cells. The DNA-induced invasion was inhibited with a broad-spectrum MMP inhibitor and partly also with chloroquine suggesting that it could be mediated via MMP activation, endosomal signaling, and TLR9. Interestingly, H. pylori DNA was shown to induce a more pronounced invasion in a gastric cancer cell line than in the other cell lines. Our results suggest that bacterial DNA as well as deoxynucleotides having stable secondary structures (i.e. hairpins or G-quadruplex structures) may serve as endogenous, invasion-inducing TLR9-ligands and promote local progression and metastasis of cancers in the alimentary tract.

Increased Toll-like receptor 9 expression indicates adverse prognosis in oesophageal adenocarcinoma.

AIMS: Toll-like receptor 9 (TLR-9) is a cellular DNA receptor that has been linked previously to invasion in various cancers. The aim of this study was to investigate TLR-9 expression and its possible association with prognosis in oesophageal adenocarcinoma. METHODS AND RESULTS: Immunohistochemical TLR-9 expression was graded in clinical specimens (n = 76) of oesophageal adenocarcinoma. The TLR-9 immunostaining intensity was compared with tumour grade, stage and indicators of proliferation, apoptosis and tumour vascular supply. High TLR-9 expression correlated with advanced tumour stage, tumour unresectability, poor differentiation and high proliferation. Strong immunoreactivity of TLR-9 also indicated poor overall survival. CONCLUSIONS: High TLR-9 expression is associated with poor differentiation, a high proliferation rate and disseminated disease. Accordingly, increased TLR-9 expression may contribute to the growth and metastatic properties of oesophageal adenocarcinoma.

Toll-like receptor 9 is a novel biomarker for esophageal squamous cell dysplasia and squamous cell carcinoma progression.

BACKGROUND: Stimulation of Toll-like receptor 9 (TLR9) has been linked to invasion in various cancer cells in vitro. We investigated TLR9 expression in normal, dysplastic and malignant esophageal squamous epithelium. METHODS: TLR9 expression was analyzed by immunohistochemistry in 46 cases of esophageal squamous cell carcinoma, including 12 cases with adjacent squamous dysplasia and 24 cases with normal esophageal epithelium. TLR9 expression was compared with tumor grade, stage, proliferation, apoptosis and vascular density. RESULTS: In normal esophageal squamous epithelium, TLR9 staining intensity decreased linearly from the basal layers to the superficial layers (p < 0.001). Strong TLR9 expression was detected across full thickness of high-grade dysplasia, the intensity clearly differing from the normal squamous epithelium and squamous cell carcinoma (p < 0.001). All squamous cell carcinomas exhibited TLR9 expression that was positively associated with a high grade (p < 0.05), the presence of lymph node metastases (p < 0.05) and previously undetected distant metastases (p < 0.05). CONCLUSIONS: Expression of TLR9 in the basal parts of normal esophageal epithelium suggests a role related to cell proliferation and differentation. TLR9 upregulation detected in dysplastic epithelium and in disseminated carcinomas indicates that this protein may serve as a novel marker for esophageal squamous dysplasia and carcinoma with metastatic potential.