Circulating microRNA-1290 as a novel diagnostic and prognostic biomarker in human colorectal cancer.

BACKGROUND: Circulating microRNAs (miRNAs) are attracting major interest as potential non-invasive biomarkers for colorectal cancer (CRC). This study aimed to identify a novel serum miRNA biomarker for the early detection and/or evaluating prognosis of CRC patients. PATIENTS AND METHODS: Comprehensive miRNA array analysis was carried out using serum samples from patients with colorectal neoplasia and healthy controls. Next, to verify whether the candidate miRNA possessed a secretory potential, we screened miRNA expression levels in culture medium from 2 CRC cell lines, followed by serum analysis from 12 stage IV CRC, 12 adenoma, and 12 control subjects. Thereafter, we validated expression of candidate miRNAs in 179 primary CRC tissues, as well as serum samples from an independent cohort of 211 CRCs, 56 adenomas, and 57 control subjects. RESULTS: Through microarray analysis, we identified significantly higher levels of miRNA-1290 (miR-1290) in serum from patients with colorectal adenomas and cancers. We verified miR-1290 overexpression in serum of CRC patients in a training cohort. In the validation cohort, serum miR-1290 levels were significantly up-regulated in patients with colorectal adenomas (P < 0.0001) and cancers (P < 0.0001). Serum miR-1290 levels could robustly distinguish adenoma [area under the curve (AUC) = 0.718] and CRC patients (AUC = 0.830) from normal subjects. High miR-1290 expression in serum and tissue was significantly associated with tumor aggressiveness and poor prognosis. Moreover, serum miR-1290 levels were an independent prognostic factor [hazard ratio (HR) = 4.51; 95% confidence interval (CI) = 1.23-23.69; P = 0.0096] and an independent predictor for tumor recurrence (hazard ratio = 3.92; 95% confidence interval = 1.11-25.14; P = 0.032) in CRC. CONCLUSIONS: Serum miR-1290 is a novel biomarker for early detection, recurrence, and prognosis in CRC.

A New Statistical Model Identified Two-thirds of Clinical T1 Gastric Cancers as Possible Candidates for Endoscopic Treatment.

BACKGROUND: Clinical T1 gastric cancer has low metastatic potential to lymph nodes and is generally curable by local treatment. Endoscopic resection can preserve the whole stomach and does not impair the patient's quality of life; however, its indication is strictly limited to the subset of patients without nodal metastasis. The study was designed to predict reliably the patients without nodal metastasis based only on the clinical information. METHODS: We examined patients with clinical T1 disease who were treated with surgery. The clinically available information was evaluated for its ability to predict nodal metastasis by logistic regression model. Then, the predictive ability of the logistic regression model using the risk factors for nodal metastasis was evaluated by a receiver operating characteristic curve. RESULTS: A total of 511 patients were entered into this study. The clinical depth (cT1a or cT1b), maximal tumor diameter, and pathological type were confirmed to be significantly different between patients with and without nodal metastasis. The cutoff value of the tumor diameter differed depending on the histology and clinical depth: 79 mm for differentiated type and 48 mm for undifferentiated type in cT1a tumors, and 43 mm for differentiated type and 11 mm for undifferentiated type in cT1b tumors. According to these criteria, 348 of the 511 patients (68.1 %) were classified to have predictive N0 status. The negative predictive value was 95.7 % (95 % confidence interval 94.0-97.5 %). CONCLUSIONS: The predictive criteria based on the multivariate logistic model identified that almost two-thirds of the patients with clinical T1 gastric cancer were possible candidates for endoscopic treatment.

Diagnostic value of computed tomography for staging of clinical T1 gastric cancer.

BACKGROUND: T1 gastric cancer can be diagnosed only by endoscopy and is almost curable by local treatment. It has been unclear how a multidetector-row computed tomography (CT) evaluation is valuable for clinical T1 patients. METHODS: Patients with clinical T1 disease, as diagnosed by endoscopy and treated with endoscopic submucosal dissection (ESD) or surgery between October 2000 and October 2007, were examined. The efficacy of CT was evaluated by the reversal rate of endoscopic T1 by CT, the incidence of clinical M1 disease, and the accuracy of diagnosing pathological N+ disease in patients who received surgery. To confirm metachronous distant and nodal metastases, the disease-free survival (DFS) also was evaluated. RESULTS: A total of 761 patients, 236 treated by ESD and 525 treated with surgery, were examined. None of the patients had an endoscopic diagnosis of clinical T1 reversed by CT. No clinical M1 disease was found. Among the 525 patients who underwent surgery, 8 showed clinical N+ disease (1.5 %), while 47 demonstrated pathological N+ disease (8.9 %). The accuracy, sensitivity, specificity, positive predictive value, and negative predictive values were 90.3, 4.3, 98.7, 25, and 91.3 %, respectively. The 5-year DFS rate was 93.6 % (95 % confidence interval 91.4-95.8 %). CONCLUSIONS: The present study suggests that diagnostic value of CT is limited for staging of clinical T1 gastric cancer patients, because the reversal rate of endoscopic T1 by CT was very low, clinical M1 disease was rare, the diagnosis of N+ status was unreliable, and metachronous M1 and N+ findings were rare.

Functional studies for the TRAF6 mutation associated with hypohidrotic ectodermal dysplasia.

BACKGROUND: Hypohidrotic ectodermal dysplasia (HED) is a rare condition characterized by hypotrichosis, hypohidrosis and hypodontia. A de novo heterozygous mutation in the tumour necrosis factor receptor-associated factor 6 gene (TRAF6) was recently identified in a patient with HED, while functional consequences resulting from the mutation remained unknown. OBJECTIVES: To determine the mechanism by which the TRAF6 mutation results in HED. METHODS: We performed coimmunoprecipitation (co-IP) studies to determine whether the mutation would affect the interaction of TRAF6 with transforming growth factor ß-activated kinase 1 (TAK1), TAK1-binding protein 2 (TAB 2) and ectodysplasin-A receptor-associated death domain protein (EDARADD). We then performed co-IP and glutathione S-transferase-pulldown assays to determine the TRAF6 binding sequences in EDARADD. In addition, we analysed the effect of the mutant TRAF6 protein on the affinity between wild-type TRAF6 and EDARADD, as well as on EDARADD-mediated nuclear factor (NF)-κB activation. RESULTS: The mutant TRAF6 protein was capable of forming a complex with TAK1 and TAB 2 in a similar way to wild-type TRAF6. However, the mutant TRAF6 protein completely lost the affinity to EDARADD, while the wild-type TRAF6 bound to the N-terminal domain of EDARADD. Furthermore, the mutant TRAF6 inhibited the interaction between the wild-type TRAF6 and EDARADD, and also potentially reduced the EDARADD-mediated NF-κB activity. CONCLUSIONS: We conclude that the mutant TRAF6 protein shows a dominant negative effect against the wild-type TRAF6 protein, which is predicted to affect the EDARADD-mediated activation of NF-κB during the development of ectoderm-derived organs, and to lead to the HED phenotype.

Effects of L-ascorbic acid 2-phosphate magnesium salt on the properties of human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: L-Ascorbic acid 2-phosphate magnesium salt (APM) is an L-ascorbic acid (AsA) derivative developed to improve AsA stability and display effective biochemical characteristics. This study aimed to investigate the effects of APM on the functions and properties of human gingival fibroblasts with respect to the prevention of periodontal disease in comparison with those of AsA. MATERIAL AND METHODS: Human gingival fibroblasts were incubated in the presence or absence of APM or L-ascorbic acid sodium salt (AsANa). Intracellular AsA was analysed by HPLC. Collagen synthesis was measured by ELISA and real-time RT-PCR. Intracellular reactive oxygen species (ROS) induced by hydrogen peroxide (H(2)O(2)) were quantified using a fluorescence reagent, and cell damage was estimated with calcein acetoxymethyl ester. Furthermore, intracellular ROS induced by tumor necrosis factor-α (TNF-α) were quantified, and expression of TNF-α-induced interleukin-8 expression, which increases due to inflammatory reactions, was measured by ELISA and real-time RT-PCR. RESULTS: APM remarkably and continuously enhanced intracellular AsA and promoted type 1 collagen synthesis and mRNA expression. Furthermore, APM decreased cell damage through the suppression of H(2)O(2)-induced intracellular ROS and inhibited interleukin-8 production through the suppression of TNF-α-induced intracellular ROS. These effects of APM were superior to those of AsANa. CONCLUSION: These results suggest that APM is more effective than AsANa in terms of intake, collagen synthesis, decreasing cell damage and inhibiting interleukin-8 expression in human gingival fibroblasts. This suggests that local application of APM can help to prevent periodontal disease.

Ectopic ovary presenting as mesenteric abscess.

Ectopic ovary is a rare gynaecological condition that results in problems with menstruation and pregnancy and may develop into a malignant tumour. However, as the condition is often asymptomatic, diagnosis is difficult and frequently delayed. We report a case of a 42-year-old female who presented with a 10-day history of abdominal pain. The patient underwent surgery that confirmed the diagnosis of an ectopic ovary with an internal abscess. The findings of our study indicate that ectopic ovaries can present with an abscess. Ectopic ovaries should be included in the differential diagnosis of masses with internal abscesses.

Influence of salivary macromolecules and fluoride on enamel lesion remineralization in vitro.

The aim of this study was to investigate the influence of salivary macromolecules on enamel lesion remineralization in the presence or absence of fluoride. Paraffin-stimulated whole saliva was centrifuged, and the supernatant was dialyzed in 1,000 molecular-weight cutoff dialysis tubes, first against a phosphate buffer and then against a mineral solution containing Ca and phosphate. Artificial subsurface lesions of human enamel, created in pH 4.5 acetate buffer, were remineralized for 28 days in 4 remineralizing solutions: group C--mineral solution as a control; group S--mineral solution + dialyzed saliva; group F--mineral solution + 1 ppm F; group SF--mineral solution + dialyzed saliva + 1 ppm F. Changes in relative mineral concentration in the lesions were assessed by transverse microradiography. The results showed statistically significant mineral gains in the lesion body in groups C (DeltaZ = 3,254 +/- 1,562% x microm) and SF (DeltaZ = 2,973 +/- 1,349% x microm), but not in groups S (DeltaZ = 5,192 +/- 1,863% x microm) and F (DeltaZ = 4,310 +/- 1,138% x microm) compared with the baseline group (DeltaZ = 5,414 +/- 461% x microm). It was also found that the mineral density at the surface layer in group F (75.0 +/- 15.7%) was greater than that in the baseline group (30.1 +/- 12.3%) with statistical significance, but not in group SF (39.9 +/- 16.5%). It was concluded that the macromolecules inhibited lesion remineralization fundamentally but that these molecules, in the presence of fluoride, seemed to play an important role in the continuation of remineralization by reducing mineral gains at the surface layer.

Modulation of prostaglandin metabolism by K-MAP and prevention of toxic effect of cyclosporin on pancreatic islet cells.

The injection of 25 mg/kg i.p. cyclosporin (CsA) for 3 wk caused marked functional and morphological deteriorations of pancreatic islet cells in Wistar rats that were prevented by the combined administration of p-aminobenzoic acid-N-D-mannoside sodium salt (K-MAP). In this article, the toxic effect of CsA on pancreatic islet cells and the preventive effect of K-MAP on CsA-associated islet cell toxicity were investigated. Prolonged hyperglycemia and depressed insulin secretion after the glucose challenge observed in CsA-treated rats could be prevented by the combined administration of 300 and 900 mg/kg K-MAP. Cytoplasmic vacuolizations and a decrease in the number of mitochondria, intact endoplasmic reticula, secretory granules, and insulin-positive cells, as revealed by peroxidase-antiperoxidase staining, could also be prevented by the administration of 900 mg/kg K-MAP. This preventive effect of K-MAP on CsA-associated islet cell toxicity may suggest the combined use of K-MAP with CsA in pancreas transplantation and treatment of insulin-dependent diabetes.

Coagulation activity is increased in the left atrium of patients with mitral stenosis.

OBJECTIVES: This study investigated the hemostatic status of the right and left atria in patients with mitral stenosis. BACKGROUND: Systemic thromboembolism is a serious major complication in patients with mitral stenosis. However, the pathogenesis of thromboembolism in mitral stenosis is not fully understood. METHODS: We determined the plasma levels of biochemical markers for platelet activity (platelet factor 4 and beta-thromboglobulin) and status of thrombin generation (fibrinopeptide A and thrombin-antithrombin III complex) and fibrinolysis (D-dimer and plasmin-alpha 2-plasmin inhibitor complex) in specimens of blood obtained from the peripheral vein and right and left atria of 12 consecutive patients with mitral stenosis who were undergoing percutaneous mitral valvuloplasty. RESULTS: Plasma levels of platelet factor 4, beta-thromboglobulin, D-dimer and plasmin-alpha 2-plasmin inhibitor complex in the patients did not differ significantly between the right and left atria, whereas levels of fibrinopeptide A and thrombin-antithrombin III complex in the left atrium were significantly higher than those in the right atrium (fibrinopeptide A in the left and right atria 19.35 +/- 4.64 and 6.31 +/- 0.75 ng/ml [mean +/- SE], respectively, p < 0.02; thrombin-antithrombin III complex in the left and right atria 11.45 +/- 2.29 and 3.98 +/- 0.60 ng/ml, respectively, p < 0.01). Levels of fibrinopeptide A and thrombin-antithrombin III complex in the left atrium did not correlate with mean transmitral gradient, dimension of the left atrium or reciprocal of the mitral valve area. Peripheral blood plasma levels of von Willebrand factor antigen were significantly higher in the patients than in an age-matched control group of normal subjects (168 +/- 25% and 99 +/- 7%, respectively, p < 0.05) but showed no difference in the peripheral blood and right and left atria of the patients. CONCLUSIONS: The coagulation system is activated in the left atrium of patients with mitral stenosis even during anticoagulation.

Expression of vascular endothelial growth factor in patients with acute myocardial infarction.

OBJECTIVE: The purpose of this study was to investigate the clinical significance of vascular endothelial growth factor (VEGF) in acute myocardial infarction (AMI). We also examined the involvement of peripheral blood mononuclear cells (PBMCs), which are a possible source of VEGF in AMI. BACKGROUND: VEGF is a potent endothelial cell-specific mitogen and could affect the outcome of AMI. METHODS: Thirty patients with AMI were used for this study. Serum and PBMCs were isolated from peripheral blood on days 1, 7, 14 and 21 after the onset of AMI. PBMCs were cultured at a density of 5 x 10(6) cells/ml for 24 h. VEGF levels in serum and the culture media were measured by enzyme-linked immunosorbent assay using a specific anti-human VEGF antibody. RESULTS: Serum VEGF levels elevated gradually after the onset of AMI and reached a peak on day 14. VEGF levels in the culture medium of PBMCs after incubation for 24 h (PBMC-VEGF) were maximally elevated 7 days after the onset. Maximum serum VEGF levels showed significant positive correlations with maximum creatine phosphokinase (CPK) levels (r = +0.70, p < 0.001), but maximum PBMC-VEGF levels did not correlate with maximum CPK levels. Patients showing improvement in left ventricular systolic function during the course of AMI showed significantly higher PBMC-VEGF levels than patients without improvement. CONCLUSIONS: The extent of myocardial damage contributes to the elevation of serum VEGF levels in AMI. VEGF produced by PBMCs may play an important role in the improvement of left ventricular function by promoting angiogenesis and reendothelialization after AMI.

Endothelin-1 production in coronary circulation in a new canine model of coronary thrombosis.

OBJECTIVE: The purpose of this study was to investigate whether endogenous endothelin-1 (ET-1) production in coronary circulation is associated with acute coronary thrombotic events in vivo. To achieve this goal, we have designed a new experimental canine model of coronary thrombosis. METHODS: In vivo occlusive thrombus was induced by the intracoronary application of radiofrequency energy (660 kHz, 50 W) in closed-chest dogs. Pathological and immunohistochemical examinations of thrombosed coronary artery were performed. In 12 dogs, plasminogen activator was administered intravenously and serial measurements of ET-1, thromboxane B2 (TXB2) and thrombin-antithrombin III complex (TAT) levels in plasma from the coronary sinus, aortic root and inferior vena cava were examined. RESULTS: Occlusive platelet-rich thrombi were attached to the deeply injured intimal surface. TAT and TXB2 increased rapidly soon after the intimal injury and declined after successful thrombolysis. In contrast, ET-1 in the coronary sinus was elevated after reperfusion and was significantly higher than in the aorta. Net ET-1 production in the coronary circulation showed a significant positive correlation with the peak TAT levels (r = 0.69, P < 0.05), but not with TXB2 or total occlusion time as an index of ischemic severity. CONCLUSIONS: Deep intimal injury leads to occlusive coronary thrombus. Thrombus formation and its subsequent lysis is associated with the activation and deactivation, respectively, of the coagulation cascade and platelets. Thrombin generation may stimulate ET-1 production in the coronary endothelium in acute coronary thrombotic events.

Coexistence of autoantibody to human thyrotropin (TSH) and autoanti-idiotypic antibody to antihuman TSH antibody in a case with simple goiter.

A 70-yr-old woman with simple goiter showed normal serum levels of T4, T3, free T4, TSH receptor antibody (TRAb) and increased TBG. Discrepancy in serum hTSH level was observed by different assay methods. Coexistence of both autoantibodies for hTSH and for anti-hTSH antibody were demonstrated by the reaction of the patient's antibody with both 125I-hTSH and 125I-anti-hTSH (monoclonal antibody; mAb). These two autoantibodies belong to the polyclonal immunoglobulin G (IgG). The autoantibody for hTSH recognized only beta-subunit of hTSH. Neither stimulating type of TRAb in Graves' disease nor blocking type of TRAb in primary hypothyroidism interfered with the binding of the patient's antibody to 125I-hTSH or 125I-anti-hTSH. Anti-idiotypic antibody (anti-ID antibody) for anti-hTSH antibody was purified by anti-hTSH antibody affinity chromatography. The binding reaction of 125I-anti-hTSH (mAb) with this anti-ID antibody could be inhibited by the unlabeled hTSH. This anti-ID antibody might represent the internal image of the nonbiological active site of TSH molecule, because of absence of thyroid stimulating activity. Goiter in this patient may have occurred by the unbound TSH with IgG (free TSH) and the bound TSH with IgG, because TSH levels in both the whole serum and the IgG free serum (the unbound TSH with IgG) were decreased significantly by T4 treatment. Coexistence of these antibodies may participate in the autoimmune mechanism of an idiotype-anti-idiotype network.

Activation of protein kinases in canine basilar artery in vasospasm.

Subarachnoid hemorrhage (SAH) often leads to a long-term narrowing of cerebra! artery called vasospasm. To understand the molecular mechanisms in vasospasm, signal transduction of tyrosine kinase pathway and phosphorylation of myosin light chain (MLC) and calponin (CaP) in the basilar artery were studied. Vasospasm was produced in the canine basilar artery by a two-hemorrhage method, and vasocontraction was induced by a local application of KCI or serotonin to the basilar artery after a transclival exposure. Intracellular substrates of tyrosine kinase pathway, including Shc, Rafl, and extracellular-regulated kinases in the basilar artery, were activated after SAH, and the activation of Shc suggests stimulation of signal transductions from tyrosine kinase receptors, G-coupled receptors, or both. The activation of tyrosine kinase pathway in vasospasm also was supported by dose-dependent dilation of the spastic basilar artery on days 0 and 7 by topical application of genistein, a tyrosine kinase inhibitor, and associated marked inhibition of tyrosine phosphorylation of intracellular substrates, including Shc. In addition, the generation of protein kinase M, catalytic fragment of protein kinase C(alpha) (PKC alpha), in vasospasm on days 0 and 7 was inhibited in response to genistein, indicating an inactivation of mu-calpain. It is suggested, therefore, that the reversal of vasospasm by genistein is closely associated with the restoration of intracellular Ca2+ levels. However, the increased activities of Raf1 and extracellular-regulated kinases in vasospasm were declined on day 7 compared with those on day 0 or 2, suggesting that the activation of tyrosine kinase pathway is more closely associated with the early stage of vasospasm than with the late stage of vasospasm. The analysis by pyrophosphate polyacrylamide gel electrophoresis (PPi-PAGE) demonstrated three MLC bands in vasospasm on days 2 and 7, as well as in KCI- and serotonin-induced vasocontraction. Since PPi-PAGE resolves smooth muscle MLC into three bands in the MLC kinase (MLCK)-mediated phosphorylation and into a single band in the PKC-mediated phosphorylation based on the phosphorylation state, the current results suggest that MLC in vasospasm is phosphorylated by MLCK but not by PKC. In basilar artery, CaP was significantly down-regulated, and in addition, significantly phosphorylated on serine and threonine residues only in vasospasm on days 2 and 7. Although the significance of CaP phosphorylations in vivo still is controversial, CaP down-regulation and phosphorylation may attenuate the inhibition of Mg(2+)-ATPase activity by CaP and induce a potential enhancement of smooth muscle contractility in delayed vasospasm. Since CaP is phosphorylated in vivo by PKC, activated PKC in vasospasm may phosphorylate CaP. Thus, SAH stimulates tyrosine kinase pathway to increase intracellular Ca2+ and activate PKC, and the former activates MLCK to phosphorylate MLC, whereas the latter phosphorylates CaP but not MLC.