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1.  High Reproducibility of Adhesion Formation in Rat with Meso-Stitch Approximation of Injured Cecum and Abdominal Wall 
Objective: Peritoneal adhesions following surgery are a common, serious pathology with severe complications. Appropriate animal adhesion models are essential for the assessment of adhesion preventing medical devices. This study introduces a variation of an established rat model in which highest degree adhesions are induced with excellent reproducibility (OPAM = optimized peritoneal adhesion model). Thus, this model seems to be eligible to study effects of adhesion preventing devices.
Methods: 24 Lewis male rats were divided into four groups (OPAM, WSFX, sham-OPAM, sham-WSFX). The OPAM technique comprised cecal abrasion, creation of an abdominal wall defect plus approximation of injured areas by a suture, which was compared to a setting of lesions without suture fixation (WSFX). All rats were sacrificed at day 7. Macroscopic and histopathological evaluations were performed. Results were statistically analyzed using ANOVA and Dunnett's test.
Results: In OPAM rats macroscopic analyses revealed a 90% incidence adhesion of cecum to the abdominal wall, all adhesions imposing as complete agglutination. In WSFX animals incidence of adhesions formation was 75%, while in both sham groups there were no adhesions at all. Histology showed the structure of adhesions with merged smooth muscle of colon and skeletal muscle of abdominal wall in all cases.
Conclusion: OPAM technique provides adhesions of injured areas with a better probability than with conventional methods. All OPAM adhesions impressed as highest degree adhesions, i.e. agglutination. Due to high reproducibility in incidence and extend of adhesion formation, the OPAM is recommended for testing of adhesion prevention medical devices.
doi:10.7150/ijms.8870
PMCID: PMC4278869  PMID: 25552912
Adhesion; Prevention; Rat; Cecal Abrasion; OPAM.
2.  The Expression Levels of Transcription Factors T-bet, GATA-3, RORγt and FOXP3 in Peripheral Blood Lymphocyte (PBL) of Patients with Liver Cancer and their Significance 
Objectives: To investigate the expression of transcriptional factors (TFs) T-bet, GATA-3, RORγt and FOXP in peripheral blood mononuclear cells (PBMC) of patients with hepatocellular carcinoma (HCC) and to evaluate the correlation between the imbalances of Th1/Th2, Th17/Treg at the expression levels and liver cancer
Methods: The peripheral venous blood was drawn from 20 HCC-patients (HCC-group) and 20 health participants (C-group). The expression levels of Th1, Th2 and Th17 and the major Treg-specific TFs T-bet, GATA-3, RORγt and FOXP3 in the PBMC were measured with quantitative real-time PCR(RT-qPCR).
Results: The mRNA level of Th1-specific TF T-bet in HCC-group was significantly lower than that of C-group (52.34±34.07 VS 104.01±56.00, P<0.01); the mRNA level of Th2-specifc TF, GATA-3, in HCC group was significantly higher than that in C-group (1.38±1.15 VS 0.58±0.65, P<0.05) and T-bet mRNA/GATA-3 mRNA ratio was significantly lower in HCC-group than in C-group (86.01±116.71 VS 461.88±708.81, P<0.05). The mRNA level of Th17-specific TF RORγt in HCC-group was significantly higher than that of C-group (72.32±32.82 VS 33.07±22.86, P<0.01). Treg-specific TF FOXP3 mRNA level was significant higher in HCC-group than in C-group (3.17±1.59 VS 1.39±1.13, P<0.01)
Conclusion: T-bet mRNA level was reduced whereas GATA-3 mRNA level was increased and T-bet/GATA-3 ratio was significantly reduced in PBMC, indicating that Th1/Th2 ratio was of imbalance at TF levels in PBMC of HCC, displaying Th2 thrift phenomena. The mRNA levels of RORγt and FOXP3 in PBMC of HCC were significantly increased, indicating the existence of a predominant phenomenon of Th17- and Treg-expressing PBMC in HCC.
doi:10.7150/ijms.8352
PMCID: PMC4278870  PMID: 25552913
RORγt; Th1; Th2; Th17; Treg; Hepatocellular carcinoma (HCC).
3.  Quantitative Detection of Circulating Nucleophosmin Mutations DNA in the Plasma of Patients with Acute Myeloid Leukemia 
Objective: The aim of this study was to quantify the copies of circulating nucleophosmin (NPM) mutations DNA in the plasma of patients with acute myeloid leukemia (AML) and to explore the association of circulating NPM mutation levels with clinical characteristics.
Design and Methods: The presence of NPM mutations in 100 Chinese patients newly diagnosed with AML were identified by RT-PCR and sequencing analysis. Copies of circulating NPM mutation A (NPM mut.A) DNA in the plasma of mutation-positive cases were quantified by real-time quantitative PCR (qRT-PCR). Furthermore, the association of circulating NPM mutation levels and clinical characteristics was analyzed.
Results: NPM mutations were identified in 37 of the 100 patients and all cases were NPM mut.A. The circulating NPM mut.A levels ranged from 0.35×108 copies/ml to 6.0×108 copies/ml in the 37 mutation-positive cases. The medium and quartile M (P25, P75) of the circulating NPM mut.A levels in patients classified as M2, M4 and M5 morphological subtypes were 1.35×108 (0.76×108, 1.91×108) copies/ml, 1.81×108 (1.47×108, 2.2×108) copies/ml and 2.50×108 (2.42×108, 3.05×108) copies/ml, respectively. Circulating NPM mut.A levels were significantly higher in patients with the M5 subtype of AML compared to patients with the M2 and M4 subtypes (p=0.000, p=0.046). In addition, circulating NPM mut.A copies were significantly associated with a higher white blood cell count, platelet count and bone marrow blast percentage (p<0.05).
Conclusion: Our results suggest that circulating NPM mutations DNA assay serves as a complementary to the routine investigative protocol of NPM-mutated leukemia.
doi:10.7150/ijms.10144
PMCID: PMC4278871  PMID: 25552914
nucleophosmin; mutation; acute myeloid leukemia; circulating DNA; real-time quantitative polymerase chain reaction.
4.  Genome-based Proteomic Analysis of Lignosus rhinocerotis (Cooke) Ryvarden Sclerotium 
Lignosus rhinocerotis (Cooke) Ryvarden (Polyporales, Basidiomycota), also known as the tiger milk mushroom, has received much interest in recent years owing to its wide-range ethnobotanical uses and the recent success in its domestication. The sclerotium is the part with medicinal value. Using two-dimensional gel electrophoresis coupled with mass spectrometry analysis, a total of 16 non-redundant, major proteins were identified with high confidence level in L. rhinocerotis sclerotium based on its genome as custom mapping database. Some of these proteins, such as the putative lectins, immunomodulatory proteins, superoxide dismutase, and aegerolysin may have pharmaceutical potential; while others are involved in nutrient mobilization and the protective antioxidant mechanism in the sclerotium. The findings from this study provide a molecular basis for future research on potential pharmacologically active proteins of L. rhinocerotis.
doi:10.7150/ijms.10019
PMCID: PMC4278872  PMID: 25552915
Lignosus rhinocerotis; proteomic analysis; LC-MS; MALDI-MS; proteins.
5.  A Microsatellite Polymorphism in IGF1 Gene Promoter and Timing of Natural Menopause in Caucasian Women 
Background: Genes involved in the IGF-1 aging pathways in the human ovary can be considered strong candidates for predictors of the natural menopause timing. This study evaluates the association between a cytosine-adenine (CA) microsatellite polymorphism in the IGF1 gene promoter P1 and age at natural menopause.
Methods: Genomic DNA was extracted from the peripheral blood, PCR was performed using primers designed to amplify the polymorphic (CA)n repeat of the human IGF1 gene, an allele dose effect for the most common (CA)19 repeats allele, Cox proportional hazard regression models and the Kaplan-Meier cumulative survivorship method with the log-rank test were used to determine statistical significance of studied associations in a sample of 257 Polish women aged 40-58 years.
Results: Crude Cox proportional hazard regression analysis confirmed the association between the IGF1 gene polymorphism and the menopause timing (p=0.038). This relationship remained statistically significant after controlling for other menopause confounders in multivariate modelling. Out of the input variables, the (CA)n polymorphism in the IGF1 gene promoter, age at menarche and smoking status were independent covariates of the natural menopause timing (χ2 =12.845; df=3; p=0.034). The onset of menopause at a younger age was likely associated with the IGF1 genotype variant not carrying the (CA)19 repeats allele, menarche before the age of 12 and a current cigarette smoker status (HR=1.6).
Conclusion: This study provides evidence that a common cytosine-adenine (CA) microsatellite repeat polymorphism in the P1 promoter region of the IGF1 gene is an independent predictive factor for age at natural menopause in Caucasian women also after adjusting for other menopause covariates.
doi:10.7150/ijms.9840
PMCID: PMC4278873  PMID: 25552916
candidate gene approach; cytosine-adenine (CA) microsatellite polymorphism; IGF1 gene; age at natural menopause; age at menarche; smoking status
6.  Effect of Helixor A on Natural Killer Cell Activity in Endometriosis 
Background and Aim: NK cells are one of the major immune cells in endometriosis pathogenesis. While previous clinical studies have shown that helixor A to be an effective treatment for endometriosis, little is known about its mechanism of action, or its relationship with immune cells. The aim of this study is to investigate the effects of helixor A on Natural killer cell (NK cell) cytotoxicity in endometriosis
Materials and Methods: We performed an experimental study. Samples of peritoneal fluid were obtained from January 2011 to December 2011 from 50 women with endometriosis and 50 women with other benign ovarian cysts (control). Peritoneal fluid of normal control group and endometriosis group was collected during laparoscopy. Baseline cytotoxicity levels of NK cells were measured with the peritoneal fluid of control group and endometriosis group. Next, cytotoxicity of NK cells was evaluated before and after treatment with helixor A. NK-cell activity was determined based upon the expression of CD107a, as an activation marker.
Results: NK cells cytotoxicity was 79.38±2.13% in control cells, 75.55±2.89% in the control peritoneal fluid, 69.59±4.96% in endometriosis stage I/II endometriosis, and 63.88±5.75% in stage III/IV endometriosis. A significant difference in cytotoxicity was observed between the control cells and stage III/IV endometriosis, consistent with a significant decrease in the cytotoxicity of NK cells in advanced stages of endometriosis; these levels increased significantly after treatment with helixor A; 78.30% vs. 86.40% (p = 0.003) in stage I/II endometriosis, and 73.67% vs. 84.54% (p = 0.024) in stage III/IV. The percentage of cells expressing CD107a was increased significantly in each group after helixor A treatment; 0.59% vs. 1.10% (p = 0.002) in stage I/II endometriosis, and 0.79% vs. 1.40% (p = 0.014) in stage III/IV.
Conclusions: Helixor A directly influenced NK-cell cytotoxicity through direct induction of CD107a expression. Our results open new role of helixor A as an imune modulation therapy, or in combination with hormonal agents, for the treatment of endometriosis.
doi:10.7150/ijms.10076
PMCID: PMC4278874  PMID: 25552917
Endometriosis; Natural Killer cells; Cytotoxicity; Helixor
7.  Astrocytes Protect Neurons from Aβ1-42 Peptide-Induced Neurotoxicity Increasing TFAM and PGC-1 and Decreasing PPAR-γ and SIRT-1 
One of the earliest neuropathological events in Alzheimer's disease is accumulation of astrocytes at sites of Aβ1-42 depositions. Our results indicate that Aβ1-42 toxic peptide increases lipid peroxidation, apoptosis and cell death in neurons but not in astrocytes in primary culture. Aβ1-42-induced deleterious neuronal effects are not present when neurons and astrocytes are mixed cultured. Stimulation of astrocytes with toxic Aβ1-42 peptide increased p-65 and decreased IκB resulting in inflammatory process. In astrocytes Aβ1-42 decreases protein expressions of sirtuin 1 (SIRT-1) and peroxisome proliferator-activated receptor γ (PPAR-γ) and over-expresses peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1) and mitochondrial transcription factor A (TFAM), protecting mitochondria against Aβ1-42-induced damage and promoting mitochondrial biogenesis.
In summary our data suggest that astrocytes may have a key role in protecting neurons, increasing neural viability and mitochondrial biogenesis, acquiring better oxidative stress protection and perhaps modulating inflammatory processes against Aβ1-42 toxic peptide. This might be a sign of a complex epigenetic process in Alzheimer's disease development.
doi:10.7150/ijms.10035
PMCID: PMC4278875  PMID: 25552918
Alzheimer's Disease; MnSOD; PPAR-γ; TFAM; PGC-1; NF-κB.
8.  The Effect of Single Embryo Transfer on Perinatal Outcomes in Japan 
Objective: In 2007 and 2008, the Japan Society for Reproductive Medicine and the Japan Society of Obstetrics and Gynecology issued a recommendation for single embryo transfer (SET). Thereafter, SET was implemented in 73% of in vitro fertilization (IVF) cases in Japan. The purpose of this study was to evaluate the effects of compliance with the SET recommendation on perinatal outcomes.
Methods: An electronic audit of the perinatal database of the Japanese Society of Obstetrics and Gynecology was conducted from 2001 through 2010. The database comprised data of 610,726 women. Totally, 20,923 women conceived through IVF. To compare perinatal outcomes, these women were categorized into two study groups depending on whether they conceived before (2004-2005, n = 3,865) or after (2009-2010, n = 6,842) the SET recommendation statement was issued.
Results: The proportion of women who conceived through IVF increased from 1.3% in 2001 to 4.8% in 2010. Compliance with the SET recommendation led to a decrease in the incidence of twin pregnancies (33.9% versus 13%, p < 0.01), incidence of preterm delivery (odds ratio [OR]: 0.54, 95% confidence interval [CI]: 0.50-0.59), low birth weight (OR: 0.42, 95% CI: 0.39-0.45), and neonatal intensive care unit admission (OR 0.70, 95% CI 0.65-0.76), but an increase in the incidence of monochorionic twins (1.6% versus 2.5%, p < 0.01).
Conclusion: Compliance with the SET recommendation improved perinatal outcomes by reducing the incidence of twin pregnancies.
doi:10.7150/ijms.10352
PMCID: PMC4278876  PMID: 25552919
in vitro fertilization; twin pregnancy; monochorionic twin; obstetric outcome; perinatal outcome.
9.  Berberine Inhibits the Metastatic Ability of Prostate Cancer Cells by Suppressing Epithelial-to-Mesenchymal Transition (EMT)-Associated Genes with Predictive and Prognostic Relevance 
Background: Over 70% of cancer metastasis from prostate cancer develops bone metastases that are not sensitive to hormonal therapy, radiation therapy, or chemotherapy. The epithelial-to-mesenchymal transition (EMT) genetic program is implicated as a significant contributor to prostate cancer progression. As such, targeting the EMT represents an important therapeutic strategy for preventing or treating prostate cancer metastasis. Berberine is a natural alkaloid with significant antitumor activities against many types of cancer cells. In this study, we investigated the molecular mechanism by which berberine represses the metastatic potential of prostate cancer.
Methods: The effects of berberine on cell migration and invasion were determined by transwell migration assay and Matrigel invasion assay. Expressions of EMT-related genes were determined by an EMT PCR Array and a quantitative RT-PCR. The prognostic relevance of berberine's modulation of EMT-related genes in prostate cancer was evaluated using Kaplan-Meier survival analysis.
Results: Berberine exerted inhibitory effects on the migratory and invasive abilities of highly metastatic prostate cancer cells. These inhibitory effects of berberine resulted in significant repression of a panel of mesenchymal genes that regulate the developmental EMT. Among EMT-related genes downregulated by berberine, high BMP7, NODAL and Snail gene expressions of metastatic prostate cancer tissues were associated with shorter survival of prostate cancer patients and provide potential therapeutic interventions.
Conclusions: We concluded that berberine should be developed as a pharmacological agent for use in combination with other anticancer drug for treating metastatic prostate cancer.
doi:10.7150/ijms.9982
PMCID: PMC4278877  PMID: 25552920
Berberine; EMT; Prostate cancer.
10.  The Regenerative Medicine in Oral and Maxillofacial Surgery: The Most Important Innovations in the Clinical Application of Mesenchymal Stem Cells 
Regenerative medicine is an emerging field of biotechnology that combines various aspects of medicine, cell and molecular biology, materials science and bioengineering in order to regenerate, repair or replace tissues.
The oral surgery and maxillofacial surgery have a role in the treatment of traumatic or degenerative diseases that lead to a tissue loss: frequently, to rehabilitate these minuses, you should use techniques that have been improved over time. Since 1990, we started with the use of growth factors and platelet concentrates in oral and maxillofacial surgery; in the following period we start to use biomaterials, as well as several type of scaffolds and autologous tissues. The frontier of regenerative medicine nowadays is represented by the mesenchymal stem cells (MSCs): overcoming the ethical problems thanks to the use of mesenchymal stem cells from adult patient, and with the increasingly sophisticated technology to support their manipulation, MSCs are undoubtedly the future of medicine regenerative and they are showing perspectives unimaginable just a few years ago. Most recent studies are aimed to tissues regeneration using MSCs taken from sites that are even more accessible and rich in stem cells: the oral cavity turned out to be an important source of MSCs with the advantage to be easily accessible to the surgeon, thus avoiding to increase the morbidity of the patient.
The future is the regeneration of whole organs or biological systems consisting of many different tissues, starting from an initial stem cell line, perhaps using innovative scaffolds together with the nano-engineering of biological tissues.
doi:10.7150/ijms.10706
PMCID: PMC4278878  PMID: 25552921
Regenerative medicine; Mesenchymal Stem Cells; Bone regeneration; Dental Pulp Stem Cells; human Periapical Cysts Mesenchymal Stem Cells; hPCy-MSCs.
11.  Combination Analysis in Genetic Polymorphisms of Drug-Metabolizing Enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A5 in the Japanese Population 
The Cytochrome P450 is the major enzyme involved in drug metabolism. CYP enzymes are responsible for the metabolism of most clinically used drugs. Individual variability in CYP activity is one important factor that contributes to drug therapy failure. We have developed a new straightforward TaqMan PCR genotyping assay to investigate the prevalence of the most common allelic variants of polymorphic CYP enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A5 in the Japanese population. Moreover, we focused on the combination of each genotype for clinical treatment. The genotype analysis identified a total of 139 out of 483 genotype combinations of five genes in the 1,003 Japanese subjects. According to our results, most of subjects seemed to require dose modification during clinical treatment. In the near future, modifications should be considered based on the individual patient genotype of each treatment.
doi:10.7150/ijms.10263
PMCID: PMC4278879  PMID: 25552922
Cytochrome P450; TaqMan-PCR; dried saliva; Japanese population; Genetic analysis.
12.  Nerve Growth Factor Protects the Ischemic Heart via Attenuation of the Endoplasmic Reticulum Stress Induced Apoptosis by Activation of Phosphatidylinositol 3-Kinase 
Background: Increased expression of nerve growth factor (NGF) has been found in the myocardium suffered from ischemia and reperfusion (I/R). The pro-survival activity of NGF on ischemic heart has been supposed to be mediated by phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway. Endoplasmic reticulum (ER) stress, which is activated initially as a defensive response to eliminate the accumulated unfolded proteins, has shown a critical involvement in the ischemia induced myocardial apoptosis. This study was aimed to investigate whether NGF induced heart protection against I/R injury includes a mechanism of attenuation of ER stress-induced myocardial apoptosis by activation of PI3K/Akt pathway.
Methods: Isolated adult rat hearts were perfused with a Langendörff perfusion system. Hearts in the Sham group were subjected to 225 min of continuous Krebs-Henseleit buffer (KHB) perfusion without ischemia. Hearts in I/R group were perfused with KHB for a 75-min of equilibration period followed by 30 min of global ischemia and 120 min of KHB reperfusion. Hearts in the NGF group accepted 45 min of euilibration perfusion and 30 min of NGF pretreatment (with a final concentration of 100 ng/ml in the KHB) before 30 min of global ischemia and 120 min of reperfusion. Hearts in K252a and LY294002 groups were pretreated with either a TrkA inhibitor, K252a or a phosphatidyl inositol 3-kinase inhibitor, LY294002 for 30 min before NGF (100 ng/ml) administration. Cardiac hemodynamics were measured from the beginning of the perfusion. Cardiac enzymes and cardiac troponin I (cTnI) were assayed before ischemia and at the end of reperfusion. Myocardial apoptosis rate was measured by TUNEL staining, and expression of glucose-related protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), caspase-12, total- and phospho-(Ser473)-Akt were assessed by Western blot analyses.
Results: NGF pretreatment significantly improved the recovery of post-ischemia cardiac hemodynamics. Reduced creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH) activity and cTnI levels, as well as decreased myocardial apoptosis ratio were observed in the NGF group. The improvement of NGF on recovery of cardiac function and alleviation of myocardial injury were completely abolished by K252a or LY294002. GRP78, caspase-12 and CHOP were highly expressed in ischemic myocardium, while NGF significantly inhibited the overexpression of these proteins which were involved in ER stress-induced myocardial apoptosis. NGF pretreatment also induced phosphorylation of Akt. When the activation of PI3K/Akt pathway is blocked by LY294002, the NGF induced suppression of the apoptosis-related proteins expression was reversed.
Conclusions: NGF pretreatment may protect the ischemic heart via inhibition of the ER stress-induced apoptosis; this pro-survival effect is mediated by PI3K/Akt pathway.
doi:10.7150/ijms.10101
PMCID: PMC4278880  PMID: 25552923
ischemia/reperfusion injury; nerve growth factor; endoplasmic reticulum; apoptosis.
13.  Association between Statin Use and Cancer: Data Mining of a Spontaneous Reporting Database and a Claims Database 
Purpose: In recent years, the potential risk of cancer associated with statin use has been a focus of much interest. However, it remains uncertain whether statin therapy is associated with cancer risk. To examine the association between statin use and the risk of cancer, we conducted data mining using the US Food and Drug Administration (FDA) Adverse Event Reporting System (FAERS) and a large organized database of claims constructed by a database vendor (The Japan Medical Data Center Co., Ltd, Tokyo, Japan [JMDC]).
Methods: Relevant reports in the FAERS, which included data from the first quarter of 2004 through the end of 2012, were identified and analyzed. The reporting odds ratio (ROR) was used to detect spontaneous report signals and was calculated using the case/non-case method. Additionally, signals were detected via the information component (IC) using the IC025 metric. Furthermore, event sequence symmetry analysis (ESSA) was applied to identify the risk of cancer following treatment with statins over the period January 2005 to July 2013.
Results: In the FAERS database analyses, significant signals for colorectal cancer and pancreatic cancer were found for statins as a class. In the ESSA, significant associations between statin use and colorectal cancer and pancreatic cancer were found, with adjusted sequence ratios (95% confidence intervals) of 1.20 (1.08-1.34) and 1.31 (1.13-1.53), respectively, at an interval of 48 months.
Conclusions: Multi-methodological approaches using different algorithms and databases suggest that statin use is associated with an increased risk for colorectal cancer and pancreatic cancer.
doi:10.7150/ijms.10656
PMCID: PMC4323360
statin use; cancer risk; FAERS database
14.  New Developments in the Pathogenesis and Therapeutic Targeting of the IDH1 Mutation in Glioma 
In the last five years, IDH1 mutations in human malignancies have significantly shaped the diagnosis and management of cancer patients. Ongoing intense research efforts continue to alter our understanding of the role of the IDH1 mutation in tumor formation. Currently, evidence suggests the IDH1 mutation to be an early event in tumorigenesis with multiple downstream oncogenic consequences including maintenance of a hypermethylator phenotype, alterations in HIF signalling, and disruption of collagen maturation contributing to a cancer-promoting extracellular matrix. The most recent reports elucidating these mechanisms is described in this review with an emphasis on the pathogenesis of the IDH1 mutation in glioma. Conflicting findings from various studies are discussed, in order to highlight areas warranting further research. Finally, the latest progress in developing novel therapies against the IDH1 mutation is presented, including recent findings from ongoing phase 1 clinical trials and the exciting prospect of vaccine immunotherapy targeting the IDH1 mutant protein.
doi:10.7150/ijms.11047
PMCID: PMC4323358
IDH1 protein; glioma; DNA methylation; HIF1A protein; molecular targeted therapy; review
15.  Analysis of Efficacy Differences between Caudal and Lumbar Interlaminar Epidural Injections in Chronic Lumbar Axial Discogenic Pain: Local Anesthetic Alone vs. Local Combined with Steroids 
Study Design: Comparative assessment of randomized controlled trials of caudal and lumbar interlaminar epidural injections in chronic lumbar discogenic pain.
Objective: To assess the comparative efficacy of caudal and lumbar interlaminar approaches of epidural injections in managing axial or discogenic low back pain.
Summary of Background Data: Epidural injections are commonly performed utilizing either a caudal or lumbar interlaminar approach to treat chronic lumbar axial or discogenic pain, which is pain exclusive of that associated with a herniated intervertebral disc, or that is due to degeneration of the zygapophyseal joints, or due to dysfunction of the sacroiliac joints, respectively. The literature on the efficacy of epidural injections in managing chronic axial lumbar pain of presumed discogenic origin is limited.
Methods: The present analysis is based on 2 randomized controlled trials of chronic axial low back pain not caused by disc herniation, radiculitis, or facet joint pain, utilizing either a caudal or lumbar interlaminar approach, with a total of 240 patients studied, and a 24-month follow-up. Patients were assigned to receive either local anesthetic only or local anesthetic with a steroid in each 60 patient group.
Results: The primary outcome measure was significant improvement, defined as pain relief and functional status improvement of at least 50% from baseline, which was reported at 24-month follow-ups in 72% who received local anesthetic only with a lumbar interlaminar approach and 54% who received local anesthetic only with a caudal approach. In patients receiving local anesthetic with a steroid, the response rate was 67% for those who had a lumbar interlaminar approach and 68% for those who had a caudal approach at 12 months. The response was significantly better in the lumbar interlaminar group who received local anesthetic only, 77% versus 56% at 12 months and 72% versus 54% at 24 months.
Conclusion: This assessment shows that in patients with axial or discogenic pain in the lumbar spine after excluding facet joint and SI Joint pain, epidural injections of local anesthetic by the caudal or lumbar interlaminar approach may be effective in managing chronic low back pain with a potential superiority for a lumbar interlaminar approach over a caudal approach.
doi:10.7150/ijms.10870
PMCID: PMC4323359
Chronic low back pain; axial low back pain; lumbar discogenic pain; caudal epidural injections; lumbar interlaminar epidural injections.
16.  Treatment Strategies for Aneurysms Associated with Moyamoya Disease 
The treatment of aneurysms associated with moyamoya disease (MMD) is difficult for neurosurgeons, and little is known of strategy options. This report constitutes a comprehensive review of the literature. We summarize the known treatments and their clinical outcomes according to the site of the aneurysm: in major arteries, peripheral arteries, moyamoya vessels, meningeal arteries, or at the site of anastomosis. The literature review indicates that the treatment of MMD-associated aneurysms varies according to the site of the aneurysm and its hemodynamic characteristics. In particular, the treatment for basilar tip aneurysms remains challenging, since both endovascular embolization and direct clipping are difficult. The potential risk for ischemia should be considered in selecting endovascular or surgical approaches. Revascularization surgery, which is important for the treatment of MMD, also determines the clinical treatment outcome of aneurysms associated with MMD.
doi:10.7150/ijms.10837
PMCID: PMC4323361
Moyamoya disease; aneurysms; treatment
17.  Genetic Interaction Analysis of TCF7L2 for Biochemical Recurrence after Radical Prostatectomy in Localized Prostate Cancer 
Backgroud: Accumulated evidence has demonstrated a significant role of the Wnt pathway in human prostate cancer. We hypothesize that genetic variants in the Wnt pathway effector, Transcription factor 7-like 2 (TCF7L2), may influence clinical outcomes in prostate cancer.
Methods: We comprehensively selected 12 tagged single-nucleotide polymorphisms (SNPs) to capture majority of common variants across TCF7L2, and genotyped in 458 localized prostate cancer patients treated with radical prostatectomy (RP). Kaplan-Meier analysis, Cox proportional hazard model, and survival tree analyses were performed to identify significant SNPs that correlated with biochemical recurrence (BCR) after surgery.
Results: A higher-order SNP-SNP interaction profile consisting of TCF7L2 rs7094463, rs10749127, and rs11196224 was significantly associated with BCR (Ptrend = 0.001). After adjusting for possible confounders, the genetic profile remained significant (Ptrend = 0.007). None of the studied SNPs were individually associated with BCR.
Conclusions: Our results support a genetic interaction in the TCF7L2 SNPs as a predictor of disease recurrence after curative RP in localized prostate cancer patients.
doi:10.7150/ijms.10953
PMCID: PMC4323362
biochemical recurrence; prostate cancer; radical prostatectomy; single-nucleotide polymorphism; TCF7L2; Wnt pathway
18.  Establishment and Characterization of an Immortalized Human Hepatic Stellate Cell Line for Applications in Co-Culturing with Immortalized Human Hepatocytes 
Background and objective. The liver-specific functions of hepatocytes are improved by co-culturing hepatocytes with primary hepatic stellate cells (HSC). However, primary HSC have a short lifespan in vitro, which is considered a major limitation for their use in various applications. This study aimed to establish immortalized human HSC using the simian virus 40 large T antigen (SV40LT) for applications in co-culturing with hepatocytes and HSC in vitro.
Methods. Primary human HSC were transfected with a recombinant retrovirus containing SV40LT. The immortalized human HSC were characterized by analyzing their gene expression and functional characteristics. The liver-specific functions of hepatocytes were evaluated in a co-culture system incorporating immortalized human hepatocytes with HSC-Li cells.
Results. The immortalized HSC line, HSC-Li, was obtained after infection with a recombinant retrovirus containing SV40LT. The HSC-Li cells were longitudinally spindle-like and had numerous fat droplets in their cytoplasm as shown using electron microscopy. Hepatocyte growth factor (HGF), VEGF Receptor 1(Flt-1), collagen type Iα1 and Iα2 mRNA expression levels were observed in the HSC-Li cells by RT-PCR. Immunofluorescence staining showed that the HSC-Li cells were positive for α-smooth muscle actin (α-SMA), platelet-derived growth factor receptor-beta (PDGFR-β), vimentin, and SV40LT protein expression. The HSC-Li cells produced both HGF and transforming growth factor-beta1 (TGF-β1) in a time-dependent manner. Real-time PCR showed that albumin, CYP3A5, CYP2E1, and UGT2B7 mRNA expression generally increased in the co-culture system. The enzymatic activity of CYP1A2 under the co-culture conditions also generally increased as compared to the monoculture of immortalized human hepatocytes.
Conclusions. We successfully established the immortalized human HSC cell line HSC-Li. It has the specific phenotypic and functional characteristics of primary human HSC, which would be a useful tool to develop anti-fibrotic therapies. Co-culturing with the HSC-Li cells improved the liver-specific functions of hepatocytes, which may be valuable and applicable for bioartificial liver systems.
doi:10.7150/ijms.11002
PMCID: PMC4323363
human hepatic stellate cells; simian virus 40 large T antigen; immortalization; immortalized human hepatocytes; co-culture; bioartificial liver
19.  Increased Expression of Macrophage Migration Inhibitory Factor and DJ-1 contribute to Cell Invasion and Metastasis of Nasopharyngeal Carcinoma 
Background and aim: Both macrophage migration inhibitory factor (MIF) and DJ-1 protein have been shown to relate with cell invasion and metastasis in tumors. However, the role of DJ-1 in invasion and metastasis of nasopharyngeal carcinoma (NPC) and its relation to MIF expression in NPC are not fully understood. The aim of present study is to determine whether or not MIF and DJ-1 are correlated with tumor invasion and influence a worse outcome in NPC, as well as its related mechanism.
Methods: 125 cases of NPC and 45 normal tissues of nasopharynx were collected. The expression of MIF and DJ-1 in tissue microarray was evaluated by immunohistochemical staining. Correlation between immunostainings and clinicopathological parameters, as well as the follow-up data of patients, was analyzed statistically. The association of MIF and DJ-1 with cell invasion and migration in NPC cell line were evaluated by small interfering RNA (siRNA) transfection, invasion assay and Western blotting.
Results: MIF and DJ-1 staining was diffused and strong in tumor cells, whereas they were generally weaker and less common in normal lining epithelia of nasopharynx. High MIF expression in tumor cells (71.2%, 89/125 cases) were significantly associated with advanced clinical stage, lymph node metastasis, and worse prognosis of NPC patients. High expression of DJ-1 (75.2%, 94/125 cases) were closely correlated to lymph node metastasis and MIF high-expression. Only MIF high expression (P = 0.010) and lymph node metastasis (P = 0.004) emerged as strong independent prognostic factors for overall survival of NPC patients. In vitro, down-regulated expression of DJ-1 in NPC cell lines by siRNA was observed to reduce cell migration and invasion potential, however, exogenous MIF promoted cells invasion.
Conclusions: The data provided evidence that increased expression of MIF and DJ-1 induced cell invasion and metastasis of NPC, supporting the idea that MIF and DJ-1 may play important roles as regulators in the progression of NPC.
doi:10.7150/ijms.7264
PMCID: PMC3880997  PMID: 24396292
nasopharyngeal carcinoma (NPC); macrophage migration inhibitory factor (MIF); DJ-1; invasion and metastasis; prognosis
20.  Influence of Endodontic Treatment on Systemic Oxidative Stress 
Introduction: An increased production of oxidizing species related to reactive oral diseases, such as chronic apical periodontitis, could have systemic implications such as an increase in cardiovascular morbidity.
Based on this consideration, we conducted a prospective study to assess whether subjects affected by chronic periodontitis presented with higher values of oxidative stress than reference values before endodontic treatment, and whether endodontic treatment can reduce the oxidative imbalance and bring it back to normal in these subjects.
Materials and methods: The authors recruited 2 groups of patients from private studies and dental clinics: these patients were recruited randomly. The oxidative balance in both patients with chronic apical periodontitis (CAP) and healthy control patients was determined by measuring the oxidant status, using an identification of the reactive oxygen metabolites (d-ROMs) test, while the antioxidant status in these patients was determined using a biological antioxidant potential (BAP) test. Both these tests were carried on plasma samples taken from enrolled patients. Values were measured both before the endodontic treatment of the patients with chronic apical periodontitis, and 30 and 90 days after treatment, and compared to those obtained from healthy control patients.
Results: It was found that, on recruitment, the patients with chronic apical periodontitis exhibited significantly higher levels of oxidative stress than control patients, as determined by the d-ROMs and BAP tests. Furthermore, the d-ROMs test values were shown to decrease and the BAP test values to increase over time in patients with chronic apical periodontitis following endodontic therapy. As the levels of oxidative stress in these patients tended to reduce and return to normal by 90 days following treatment.
Conclusions: This study has demonstrated a positive association between chronic apical periodontitis and oxidative stress. Subjects affected by chronic apical periodontitis are exposed to a condition of oxidative stress, which is extremely dangerous to general health. Moreover, one can infer from these findings that through proper endodontic therapy, a good oxidative balance can be restored, thereby avoiding the risk of contracting the abovementioned diseases.
doi:10.7150/ijms.6663
PMCID: PMC3880985  PMID: 24396280
Oxidative stress; chronic apical periodontitis; endodontic treatment.
21.  17β-Estradiol Inhibits Mesenchymal Stem Cells-Induced Human AGS Gastric Cancer Cell Mobility via Suppression of CCL5- Src/Cas/Paxillin Signaling Pathway 
Gender differences in terms of mortality among many solid organ malignancies have been proved by epidemiological data. Estrogen has been suspected to cast a protective effect against cancer because of the lower mortality of gastric cancer in females and the benefits of hormone replacement therapy (HRT) in gastric cancer. Hence, it suggests that 17β-estradiol (E2) may affect the behavior of cancer cells. One of the key features of cancer-related mortality is metastasis. Accumulating evidences suggest that human bone marrow mesenchymal stem cells (HBMMSCs) and its secreted CCL-5 have a role in enhancing the metastatic potential of breast cancer cells. However, it is not clear whether E2 would affect HBMMSCs-induced mobility in gastric cancer cells. In this report, we show that CCL-5 secreted by HBMMSCs enhanced mobility in human AGS gastric cancer cells via activation of Src/Cas/Paxillin signaling pathway. Treatment with specific neutralizing antibody of CCL-5 significantly inhibited HBMMSCs-enhanced mobility in human AGS gastric cancer cells. We further observe that 17β-estradiol suppressed HBMMSCs-enhanced mobility by down-regulating CCL5-Src/Cas/paxillin signaling pathway in AGS cells. Collectively, these results suggest that 17β-estradiol treatment significantly inhibits HBMMSCS-induced mobility in human AGS gastric cancer cells.
doi:10.7150/ijms.6851
PMCID: PMC3880986  PMID: 24396281
Estrogen; Mesenchymal stem cell; Human gastric cancer cell; CCL-5; Cell mobility
22.  Hypoxia-Induced Deregulation of miR-126 and Its Regulative Effect on VEGF and MMP-9 Expression 
Objective: miR-126, the miRNA considered to be specially expressed in endothelial cells and hematopoietic progenitor cells, is strongly associated with angiogenesis. The purpose is to evaluate the role of miR-126 in hypoxia-induced angiogenesis and the possible mechanisms. Methods: The expression of miR-126 was detected in hypoxia-treated RF/6A cells and diabetic retinas using real-time PCR. The miR-126 was up- or down-regulated by transfecting miR-126-mimics or inhibitors into RF/6A cells. Cell cycle analysis was performed using flow cytometry. The protein levels of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) were assessed by immunoblotting. Results: A significantly decreased expression of miR-126 was found in hypoxia-treated RF/6A cells in a time-dependent manner compared with normoxic condition. The expression of miR-126 was also reduced in the retina tissue of streptozotocin-induced diabetic rats. The expression of VEGF and MMP-9 proteins was increased in hypoxia-induced RF/6A cells. In the functional analysis, miR-126-mimic significantly reduced the percentage of RF/6A cells in S phases compared with the negative control under hypoxic conditions. Furthermore, the VEGF and MMP-9 protein levels were sharply decreased in hypoxia-induced RF/6A cells pretreated with miR-126-mimics and increased in the cells pretreated with miR-126-inhibitors. Conclusions: miR-126 is down-regulated under hypoxic condition both in vitro and in vivo and may halt the hypoxia-induce neovascularization by suspending the cell cycle progression and inhibiting the expression of VEGF and MMP-9.
doi:10.7150/ijms.7329
PMCID: PMC3880987  PMID: 24396282
MicroRNA-126; Hypoxia; Vascular endothelial growth factor; Matrix metalloproteinase-9; Diabetic retinopathy.
23.  Differential Protein Expression between Chondrogenic Differentiated MSCs, Undifferentiated MSCs and Adult Chondroctyes Derived from Oryctolagus cuniculus in vitro 
Objective: This preliminary study aims to determine the differentially expressed proteins from chondrogenic differentiated multipotent stromal cells (cMSCs) in comparison to undifferentiated multipotent stromal cells (MSCs) and adult chondrocytes (ACs).
Methods: ACs and bone marrow-derived MSCs were harvested from New Zealand White rabbits (n = 3). ACs and cMSCs were embedded in alginate and were cultured using a defined chondrogenic medium containing transforming growth factor-beta 3 (TGF-β3). Chondrogenic expression was determined using type-II collagen, Safranin-O staining and glycosaminoglycan analyses. Two-dimensional gel electrophoresis (2-DE) was used to isolate proteins from MSCs, cMSCs and ACs before being identified using liquid chromatography-mass spectrometry (LC-MS). The differentially expressed proteins were then analyzed using image analysis software.
Results: Both cMSCs and ACs were positively stained with type-II collagen and safranin-O. The expression of glycosaminoglycan in cMSCs was comparable to AC at which the highest level was observed at day-21 (p>0.05). Six protein spots were found to be most differentially expressed between MSCs, cMSCs and ACs. The protein spots cofilin-1 (CFL1) and glycealdehyde-3-phosphate dehydrogenase (GAPD) from cMSCs had expression levels similar to that of ACs whereas the others (ie. MYL6B, ALDOA, TAGLN2, EF1-alpha), did not match the expression level of ACs.
Conclusion: Despite having similar phenotypic expressions to ACs, cMSCs expressed proteins which were not typically expected. This may explain the reason for the unexplained lack of improvement in cartilage repair outcomes reported in previous studies.
doi:10.7150/ijms.7244
PMCID: PMC3880988  PMID: 24396283
2D gel electrophoresis; Chondrocytes; Chondrogenic multipotent stromal cells; Chondrogenesis; LC-MS; Multipotent mesenchymal stem cells; Protein expression; orthopaedics
24.  A Comparative Clinical Study on Five Types of Compression Therapy in Patients with Venous Leg Ulcers 
The aim of this study was to compare five types of compression therapy in venous leg ulcers (intermittent pneumatic vs. stockings vs. multi layer vs. two layer short stretch bandages vs. Unna boots). Primary study endpoints were analysis of changes of the total ulcer surface area, volume and linear dimensions inside observed groups. The secondary end points were comparisons between all groups the number of completely healed wounds (ulcer healing rates), Gilman index and percentage change of ulcer surface area. In total, 147 patients with unilateral venous leg ulcers were included to this study. Participants were randomly allocated to the groups: A, B, C, D and E. After two months the healing rate was the highest in group A (intermittent pneumatic compression) - 57.14%, 16/28 patients, B (ulcer stocking system) - 56.66%, 17/30 patients and C (multi layer short stretch bandage) - 58.62%, 17/29 patients. Significantly much worse rate found in group D (two layer short stretch bandages) - only 16.66%, 5/30 patients and E (Unna boots) - 20%, 6/30 patients. The analysis of changes of the percentage of Gilman index and wound total surface area confirmed that intermittent pneumatic compression, stockings and multi layer bandages are the most efficient. The two layer short - stretch bandages and Unna boots appeared again much less effective.
doi:10.7150/ijms.7548
PMCID: PMC3880989  PMID: 24396284
compression stockings; short stretch bandages; Unna boots; venous leg ulcers; wound healing
25.  Amelioration of Radiation-induced Skin Injury by HIV-TAT-Mediated Protein Transduction of RP-1 from Rana pleurade 
Radiation-induced reactive oxygen species (ROS) can damage DNA and most other biological macromolecules in skin and radiation-induced skin injury is a serious concern for radiation therapy. Skin possesses an extremely efficient antioxidant system, which is conferred by two systems: antioxidant enzymes and small molecules that can scavenge ROS by donating electrons. Amphibian skin is a multifunctional organ, which protects against dangers of various oxidative stresses. Recently, a small peptide called RP-1 was isolated from the skin secretions of Rana pleurade, which shows strong antioxidant activity. However, this RP-1 peptide is limited because its inability to across the cell membrane. Protein transduction domains (PTDs) have demonstrated high efficiency for facilitating the internalization of both homologous and heterogeneous proteins into cells. This study aims to elucidate the protective effects of a HIV-TAT (TAT) PTD-coupled RP-1 fusion protein (TAT-RP1) on radiation-induced skin injury in vitro and in vivo. The synthesized fusion TAT-RP1 peptide can be incorporated into human keratinocyte HaCaT cells in a dose- and time-dependent manner without cytotoxicity. We then evaluated the protective role of TAT-RP1 against ionizing radiation. TAT-RP1 supplementation increased anti-superoxide anion ability of HaCaT cells and decreased HaCaT cell radiosensitivity to irradiation. Moreover, TAT-RP1 was able to penetrate the skin of rats, entering epidermis as well as the dermis of the subcutaneous layer in skin tissue. Topical spread of TAT-RP1 promoted the amelioration of radiation-induced skin damage in rats. These results suggest that TAT-RP1 has potential as a protein therapy for radiation-induced skin injury.
doi:10.7150/ijms.7463
PMCID: PMC3880990  PMID: 24396285
protein transduction domain (PTD); HIV-TAT; RP-1; radiation-induced skin injury; HaCaT cells

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