Involvement of microRNA-124 in biological behaviors of laryngeal carcinoma via PLOD2 signaling pathway.

OBJECTIVE: The purpose of this study was to investigate the role of microRNA-124 (miR-124) in laryngeal carcinoma (LC) and to explore the underlying mechanisms. PATIENTS AND METHODS: LC tissues were collected from 98 patients diagnosed with LC in our hospital. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) analysis was used to detect the expression levels of miR-124 and procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) in LC tissues and cell lines. Bioinformatics (TargetScan and miRDB) and double Luciferase assay were performed to predict and confirm the relationship between PLOD2 and miR-124 in LC, respectively. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay and the colony formation assay were designed to measure the proliferation ability of the cells. Additionally, transwell and wound healing assays were used to explore cell migration and invasion. RESULTS: MiR-124 was found significantly downregulated both in LC tissues and cells. PLOD2 was predicted and confirmed as the target gene of miR-124 in LC. By regulating the protein expression of PLOD2, miR-124 could significantly suppress the proliferation, migration, and invasion of cells. However, the transfection of PLOD2 could partially offset the effects of miR-124 on LC cells. CONCLUSIONS: MiR-124 was involved in regulating the malignant behaviors of LC cells. Furthermore, the miR-124/PLOD2 axis might become a potential target for the treatment of LC.

The mechanism of PDX in regulating cervical cancer HeLa cell proliferation and tumor formation.

OBJECTIVE: As a gynecological malignant tumor, cervical cancer tends to occur in younger patients. Furin is an important member of precursor protein convertase that may affect malignant tumor metastasis and neovascularization. Pancreatic duodenal homeobox (PDX), as a transcription factor, can inhibit Furin. This study intends to explore the impact of PDX on cervical cancer HeLa cell proliferation and mechanism. PATIENTS AND METHODS: PDX plasmid was transfected to cervical cancer HeLa cells. Cell proliferation, invasion, and migration were tested. HeLa cells were injected to Balb/c nude mice to observe the change of general status, tumor formation rate, tumor weight, and volume. RESULTS: PDX expression was gradually increased after PDX transfection following time extension. Cell proliferation, invasion, and migration in experimental group were significantly lower than those in normal control (p < 0.05). The nude mice injected with PDX transfected HeLa cells showed markedly better general status, with reducing rate of tumor formation, tumor weight and volume compared with control. CONCLUSIONS: PDX can suppress cervical cancer HeLa cell proliferation, cell invasion and migration, improve general status of tumor-bearing nude mice and reduce tumor weight and volume. Our data highlight the clinical benefits of PDX in inhibiting cervical cancer proliferation, invasion, and metastasis.

Factors associated with villus atrophy in symptomatic coeliac disease patients on a gluten-free diet.

BACKGROUND: Duodenal injury persists in some coeliac disease patients despite gluten-free diet, and is associated with adverse outcomes. AIM: To determine the prevalence and clinical risk factors for persistent villus atrophy among symptomatic coeliac disease patients. METHODS: A nested cross-sectional analysis was performed on coeliac disease patients with self-reported moderate or severe symptoms while following a gluten-free diet, who underwent protocol-mandated duodenal biopsy upon enrolment in the CeliAction clinical trial. Demographic factors, symptom type, medication use, and serology were examined to determine predictors of persistent villus atrophy. RESULTS: Of 1345 symptomatic patients, 511 (38%, 95% CI, 35-41%) were found to have active coeliac disease with persistent villus atrophy, defined as average villus height to crypt depth ratio ≤2.0. On multivariable analysis, older age (OR, 5.1 for ≥70 vs. 18-29 years, 95% CI, 2.5-10.4) was a risk factor while longer duration on gluten-free diet was protective (OR, 0.37, 95% CI, 0.24-0.55 for 4-5.9 vs. 1-1.9 years). Villus atrophy was associated with use of proton-pump inhibitors (PPIs; OR, 1.6, 95% CI, 1.1-2.3), non-steroidal anti-inflammatory drugs (NSAIDs; OR, 1.64, 95% CI, 1.2-2.2), and selective serotonin reuptake inhibitors (SSRIs; OR, 1.74, 95% CI, 1.2-2.5). Symptoms were not associated with villus atrophy after adjusting for covariates. Conclusions A majority of symptomatic coeliac disease patients did not have active disease on follow-up histology. Symptoms were poorly predictive of persistent mucosal injury. The impact of NSAIDs, PPIs, and SSRIs on mucosal healing in coeliac disease warrants further study.

Immunopathogenesis of olmesartan-associated enteropathy.

BACKGROUND: Olmesartan-associated enteropathy (OAE) is characterised by diarrhoea, nausea, vomiting, abdominal pain, weight loss and severe sprue-like enteropathy, all of which are resolved after discontinuation of olmesartan medoximil. AIM: To determine the mechanistic similarities of OAE with coeliac sprue. METHODS: Duodenal biopsies were extracted from OAE patients before (n = 11) or after (n = 17) discontinuation of olmesartan medoxomil (on or off olmesartan medoxomil). There were seven 'on/off' paired samples. Formalin-fixed biopsies were stained for CD8, CD4, FoxP3, IL-15R and psmad 2/3. Caco2 cells (human colonic epithelial line) were treated with olmesartan medoxomil and stained for IL-15, IL-15R and ZO-1. RESULTS: In the 'on olmesartan medoxomil' duodenal biopsies, a significant increase in the numbers of CD8+ cells and the number of cells that are FoxP3+ (a regulatory T-cell marker) are present in the duodenum as compared to the duodenal biopsies from patients who discontinued olmesartan medoxomil. IL15R expression is also increased with olmesartan medoxomil use. Evaluation of the effect of olmesartan medoxomil upon Caco-2 cells demonstrated that IL15 expression is increased in response to olmesartan medoxomil treatment. Further, ZO-1, a tight junction protein, is disrupted in olmesartan medoxomil-treated Caco-2 cells. CONCLUSIONS: Olmesartan-associated enteropathy shares many features with coeliac disease, including symptoms and immunopathogenic pathways, such as increased numbers of CD8+ cells and corresponding overexpression of IL15 by epithelial cells. Taken together, the treatment of epithelial cells with olmesartan medoxomil induces a response by intestinal epithelial cells that is similar to the innate effects of gluten upon the epithelium of coeliac patients.

Pain in patients with equal radiographic grades of osteoarthritis in both knees: the value of gray scale ultrasound.

OBJECTIVES: To investigate the association of ultrasound (US) features with pain and the functional scores in patients with equal radiographic grades of osteoarthritis (OA) in both knees. METHODS: Fifty-six consecutive patients with knee OA: 85 symptomatic knees (81 knees with medial pain) and 27 asymptomatic knees, and 10 healthy patients without knee OA as a control were enrolled. US was done by two ultrasonographers blinded to patient diagnoses. US features were semiquantitatively scored (0-3) when appropriate. RESULTS: In the OA group, common US findings were marginal osteophyte, suprapatellar synovitis, suprapatellar effusion (SPE), medial meniscus protrusion, medial compartment synovitis (MCS), lateral compartment synovitis, and Baker's cyst. Only SPE and MCS were significantly associated with knee pain. Visual analog pain scale (VAS) scores on motion were positively linearly associated with SPE and MCS (P < 0.01). Only MCS was degree-dependently associated with VAS scores at rest, the Western Ontario and McMaster Universities pain subscale, and the presence of medial knee pain (P < 0.01) after adjustments for age, gender, body mass index (BMI), radiographic grade, and other US features. In the control group, no US features were associated with knee pain. CONCLUSIONS: US inflammation features, including SPE and MCS, were positively linearly associated with knee pain in motion. MCS was also degree-dependently associated with pain at rest and the presence of medial knee pain. These findings show that synovitis was one important predictive factor of pain. Further studies to confirm the association of US features and pain are warranted.

Analyses of PRMT1 proteins in human colon tissues from Hirschsprung disease patients.

BACKGROUND: Protein arginine methyltransferase 1 (PRMT1) catalyzes the majority of arginine methylation in cells and plays important roles in the differentiation and development of neurons. It is also implicated in the regulation of nitric oxide synthetase (NOS). Hirschsprung disease (HSCR) is characterized by the absence of intramural ganglion cells in the nerve plexuses of the distal gut. METHODS: Western blot analyses revealed reduced PRMT1 protein levels in the aganglionosis segments of HSCR patients. Immunohistochemistry detected PRMT1 expression in the colonic mucosa, the enteric nervous system (ENS) and endothelial cells. Specific and strong PRMT1 expression in neuron cell bodies of the plexus was demonstrated by immunofluorescent double-labeling with neuron-specific marker HuC/D. KEY RESULTS: In the mucosa, PRMT1 was detected at all crypt cells. Intensive PRMT1 staining was detected in the submucosal and the myenteric plexuses in normal or oligoganglionosis segments. Aganglionosis segments from HSCR patients contain no plexuses, and thus not labeled with PRMT1. The phenomenon is specific to the megacolon of HSCR as strong PRMT1 staining was observed in plexuses of the rectal ectasia segments (dilated rectum and distal sigmoid not related with aganglionosis) from anorectal malformation patients. Furthermore, PRMT1 was also present in the same neuronal cells expressing neuronal NOS in the plexuses. CONCLUSIONS & INFERENCES: We suggest that PRMT1 can be a useful marker for HSCR. This study is the first illustration of PRMT1 protein expression in human tissues from non-cancerous disease and set up the base for further investigations of PRMT1 function in ENS development and intestinal motility.

Intracameral recombinant tissue plasminogen activator for the treatment of severe fibrin reaction in endophthalmitis.

PURPOSE: The authors sought to evaluate the efficacy of recombinant tissue plasminogen activator (r-TPA) in the treatment of severe fibrinous anterior chamber reactions secondary to endophthalmitis. METHODS: Twelve patients with endophthalmitis associated with severe fibrinous anterior chamber reactions were enrolled in the study. Various degrees of posterior synechiae were noted in 10 of these patients. TPA (25 microg/0.05 cc) was injected into the anterior chamber through the limbus as an adjunctive treatment to intravitreal antibiotic injection. Efficacy of the treatment was judged by the rate of fibrinolysis, the lysis of posterior synechiae, and the size of the dilated pupil. RESULTS: After application of 25 microg of r-TPA, the fibrin reaction gradually resolved in 2-14 h. The median dilated pupil size in ratio 24 h after r-TPA injection was significantly larger than before r-TPA injection (0.41 vs 0.60; P=0.002). The median difference in pupil size in ratio in patients with posterior synechiae larger than 180 degrees was significantly larger than those with posterior synechiae equal or less than 180 degrees (0.32 vs 0.09; P=0.003). At 24 h after application of r-TPA, no eye had posterior synechiae. CONCLUSION: Intracameral injection of r-TPA may be a safe and effective method for the treatment of significant fibrin reaction in endophthalmitis and thus facilitates vitreous and fundus examinations and vitrectomy if necessary.

Therapy-induced expression of NF-kappaB portends poor prognosis in patients with localized esophageal cancer undergoing preoperative chemoradiation.

Activated nuclear factor-kappa B (NF-kappaB) in the pretreatment cancer tissue of patients with localized esophageal adenocarcinoma (LEA) undergoing preoperative chemoradiation is associated with poor prognosis. It is known that constitutively activated NF-kappaB prior to any therapy portends poor prognosis, and it is also known that activated NF-kappaB in the treated specimen is associated with poor prognosis. However, the prognosis of patients who have treatment-induced activation of NF-kappaB (meaning their cancers activate NF-kappaB during or after therapy) is not been reported. We hypothesized that the treatment-induced activation of NF-kappaB would impart poor prognosis similar to that imparted by constitutively activated NF-kappaB cancer. Patients with LEA who had undergone preoperative chemoradiation plus surgery and had pre- and post-therapy cancer tissue available were selected. Pre- and post-therapy cancer tissues were stained by immunohistochemistry for nuclear staining of NF-kappaB. The overall survival (OS) and disease-free survival were assessed and compared for patients who had intrinsic constitutively activated NF-kappaB cancer with those who had induced activation of NF-kappaB only post-therapy. A total of 41 patients with LEA were investigated. Twenty-five patients had NF-kappaB positive cancer at baseline, and 16 had NF-kappaB negative cancer at baseline but became positive post-therapy. There was no difference in the location, histology grade, clinical stage, or the curative resection (RO) resection rate in the two populations. OS (P = 0.71), disease-free survival (P = 0.86), and median survivals (Converters: 24 months [95% confidence intervals: 7.78 to 40.22]vs. Nonconverters: 34.13 months [95% confidence intervals: 3.54 to 64.27]) were not different between the two groups. Our data suggest that activation of NF-kappaB in response to stress/injury of therapy leads to poor OS. These results need to be confirmed in a larger number of patients. It may be that only pre-therapy evaluation of NF-kappaB is insufficient to assess prognosis of patients with LEA. Additional implications include that when effective anti-NF-kappaB therapies become available, they may have to be considered in patients whose cancers do not have constitutively activated NF-kappaB or cancer may have to be monitored during therapy with biomarker assessments.

Apoptotic insults to human HepG2 cells induced by S-(+)-ketamine occurs through activation of a Bax-mitochondria-caspase protease pathway.

BACKGROUND: Ketamine is widely used as an i.v. anaesthetic agent and as a drug of abuse. Hepatocytes contribute to the metabolism of endogenous and exogenous substances. This study evaluated the toxic effects of S-(+)-ketamine and possible mechanisms using human hepatoma HepG2 cells as the experimental model. METHODS: HepG2 cells were exposed to S-(+)-ketamine. Cell viability and the release of lactate dehydrogenase (LDH) and gamma-glutamyl transpeptidase (GPT) were measured to determine the toxicity of S-(+)-ketamine to HepG2 cells. Cell morphology, DNA fragmentation, and apoptotic cells were analysed to evaluate the mechanism of S-(+)-ketamine-induced cell death. Amounts of Bax, an apoptotic protein, and cytochrome c in the cytoplasm or mitochondria were quantified by immunoblotting. Cellular adenosine triphosphate levels were analysed using a bioluminescence assay. Caspases-3, -9, and -6 were measured fluorometrically. RESULTS: Exposure of HepG2 cells to S-(+)-ketamine increased the release of LDH and GPT, but decreased cell viability (all P<0.01). S-(+)-Ketamine time-dependently caused shrinkage of HepG2 cells. Exposure to S-(+)-ketamine led to significant DNA fragmentation and cell apoptosis (P=0.003 and 0.002). S-(+)-Ketamine increased translocation of Bax from the cytoplasm to mitochondria, but decreased the mitochondrial membrane potential and cellular adenosine triphosphate levels (all P<0.01). Sequentially, cytosolic cytochrome c levels and activities of caspases-9, -3, and -6 were augmented after S-(+)-ketamine administration (all P<0.001). Z-VEID-FMK, an inhibitor of caspase-6, alleviated the S-(+)-ketamine-induced augmentation of caspase-6 activity, DNA fragmentation, and cell apoptosis (all P<0.001). CONCLUSIONS: This study shows that S-(+)-ketamine can induce apoptotic insults to human HepG2 cells via a Bax-mitochondria-caspase protease pathway. Thus, we suggest that S-(+)-ketamine at a clinically relevant or an abused concentration may induce liver dysfunction possibly due to its toxicity to hepatocytes.

Significance of post-chemoradiation biopsy in predicting residual esophageal carcinoma in the surgical specimen.

Pathologic complete response in the resected esophagus can be achieved in approximately 30% of patients with locally advanced esophageal or gastroesophageal junction carcinoma after preoperative chemoradiation therapy. These patients tend to have a longer survival than those who have less than pathologic complete response. Post-chemoradiation esophageal biopsy (PCEB) is used to check for the presence of residual tumor before a definitive resection is performed, but the clinical significance of PCEB findings is not clear due to the possibility of sampling bias and the superficial nature of the specimen obtained. We evaluated the use of PCEB (defined as biopsy taken within 30 days before esophagectomy) in predicting residual cancer in post-treatment esophagectomy specimens. PCEB was performed in 65 of 183 (36%) patients with locally advanced esophageal or gastroesophageal junction carcinoma, who received preoperative chemoradiation therapy. The cancer status in PCEB was correlated with the residual cancer in the esophagectomy specimens. PCEB had no cancer in 80% (52 of 65) of patients (Bx-negative) and cancer in 20% (13 of 65) of patients (Bx-positive). There was no difference in the presence of residual cancer (either in esophagus or lymph node) in esophagectomy specimens between Bx-negative patients (77%, 40 of 52) or Bx-positive patients (92%, 12 of 13), P = 0.44. The positive predictive value of biopsy was 92% (12 of 13), negative predictive value 23% (12 of 52), sensitivity 23% (12 of 52) and specificity 92% (12 of 13). There was no difference in the residual cancer staging in the esophagectomy specimen between Bx-positive and Bx-negative patients. In contrast, residual metastatic carcinoma in lymph nodes was more frequent in Bx-positive patients (69.2%, 9 of 13) than in Bx-negative patients (28.8%, 15 of 52), P = 0.01. Our data suggest that PCEB is a specific but not a sensitive predictor of residual cancer following esophagectomy. Bx-positive patients tend to have more frequent residual tumor in lymph nodes. The utility of PCEB in predicting residual cancer in the lymph nodes needs to be explored further along with molecular predictors of response to preoperative therapy.

Chemoprevention for Barrett's esophagus trial. Design and outcome measures.

Barrett's esophagus is a premalignant condition in which normal squamous epithelium of the esophagus is replaced by metaplastic columnar epithelium. It is a known risk factor for the development of esophageal adenocarcinoma. With the incidence of esophageal adenocarcinoma rising, it is reasonable to study Barrett's esophagus as a potential target for therapy that may prevent, delay and/or reverse ongoing tumorigenic processes. Epidemiologic and animal studies support the use of nonsteroidal anti-inflammatory drugs (NSAIDs) in the chemoprevention of several cancers, including esophageal cancer. Cyclo-oxygenase-2 (COX-2) inhibitors are a new class of NSAIDs that inhibit prostaglandin synthesis by selectively blocking the COX-2 enzyme. The COX-2 enzyme has been reported to be over-expressed in premalignant and malignant states, including in Barrett's esophagus and esophageal adenocarcinoma. The Chemoprevention for Barrett's Esophagus Trial (CBET) is a phase IIb, multicenter, randomized, double-masked, placebo-controlled study of the selective COX-2 inhibitor, celecoxib, in patients with Barrett's dysplasia. The sample size is 200 patients with high or low grade Barrett's dysplasia. Celecoxib is administered orally, 200 mg twice per day; the dosing schedule for placebo is the same. Randomization is stratified by dysplasia grade and by clinic. Endoscopy with biopsies is performed at specified time intervals according to the highest grade of dysplasia determined at randomization. The primary outcome measure is the change from baseline to 1 year in the proportion of biopsies exhibiting dysplasia. Secondary outcomes include change from baseline in the maximal grade, extent and surface area of dysplasia. Tertiary outcomes will include measurements of various relevant biomarkers.