International Society of Paediatric Oncology (SIOP) Global Mapping Programme: Latin American Society of Pediatric Oncology (SLAOP) country-level report.

BACKGROUND: Latin American countries are improving childhood cancer care, showing strong commitment to implement the Global Initiative for Childhood Cancer, but there are scant publications of the situation at a continental level. METHODS: As part of the International Society of Paediatric Oncology Global Mapping project, delegates of each country participating in the Latin American Society of Pediatric Oncology (SLAOP) and chairs of national pediatric oncology societies and cooperative groups were invited to provide information regarding availability of national pediatric cancer control programs (NPCCP), pediatric oncology laws, pediatric oncology tumor registries, and training programs and support to diagnosis and treatment. RESULTS: Nineteen of the 20 countries participating in SLAOP responded. National delegates reported nine countries with NPCCP and four of them were launched in the past 5 years. National pediatric tumor registries are available in eight countries, and three provided published survival results. Fellowship programs for training pediatric oncologists are available in 12 countries. National delegates reported that eight countries provide support to most essential diagnosis and treatments and 11 provide partial or minimal support that is supplemented by civil society organizations. Seven countries have a pediatric oncology law. There are three international cooperative groups and four national societies for pediatric oncology. CONCLUSION: Despite many challenges, there were dramatic advances in survivorship, access to treatment, and availability of NPCCP in Latin America. Countries with highest social development scores in general provide more complete support and are more likely to have NPCCP, training programs, and reported survival results.

Exchange of cellular components between platelets and tumor cells: impact on tumor cells behavior.

Background: Platelets are active players in tumorigenesis, although the exact interactive mechanisms and their direct impact on tumor cells remain largely unknown. Methods: Bidirectional transference of lipids, proteins and RNA between platelets and tumor cells and its impact on tumor cell behavior and tumor process are analyzed in this work. Phenotypic, genetic and functional modifications induced by platelets were analyzed both in tumor cell lines and in circulating tumor cells (CTCs). Results: Data from these assays showed that platelets transferred structural components to tumor cells with higher efficiency than tumor cells to platelets (p = 0.001). This biological interplay occurred by direct contact, internalization or via extracellular vesicles. As a result, tumor cells acquired platelet markers (CD61 and CD42), showed decreased EpCAM, expressed epithelial-to-mesenchymal transition markers, and increased proliferation rates. Moreover, we were able to detect CD61 in CTCs from early and advanced prostate cancer. Conclusions: Our results demonstrated, for the first time, that platelets educate tumor cells by highly efficient transference of lipids, proteins and RNA through different mechanisms. These results suggest that tumor cells and CTCs might acquire highly dynamic and aggressive phenotypes due to platelets interaction including EMT, stem-like phenotype and high proliferative rates.

Cross-Resistance to Abiraterone and Enzalutamide in Castration Resistance Prostate Cancer Cellular Models Is Mediated by AR Transcriptional Reactivation.

Androgen deprivation therapy (ADT) and novel hormonal agents (NHAs) (Abiraterone and Enzalutamide) are the goal standard for metastatic prostate cancer (PCa) treatment. Although ADT is initially effective, a subsequent castration resistance status (CRPC) is commonly developed. The expression of androgen receptor (AR) alternative splicing isoforms (AR-V7 and AR-V9) has been associated to CRPC. However, resistance mechanisms to novel NHAs are not yet well understood. Androgen-dependent PCa cell lines were used to generate resistant models to ADT only or in combination with Abiraterone and/or Enzalutamide (concomitant models). Functional and genetic analyses were performed for each resistance model by real-time cell monitoring assays, flow cytometry and RT-qPCR. In androgen-dependent PCa cells, the administration of Abiraterone and/or Enzalutamide as first-line treatment involved a critical inhibition of AR activity associated with a significant cell growth inhibition. Genetic analyses on ADT-resistant PCa cell lines showed that the CRPC phenotype was accompanied by overexpression of AR full-length and AR target genes, but not necessarily AR-V7 and/or AR-V9 isoforms. These ADT resistant cell lines showed higher proliferation rates, migration and invasion abilities. Importantly, ADT resistance induced cross-resistance to Abiraterone and/or Enzalutamide. Similarly, concomitant models possessed an elevated expression of AR full-length and proliferation rates and acquired cross-resistance to its alternative NHA as second-line treatment.

Sandwich-Type Electrochemical Paper-Based Immunosensor for Claudin 7 and CD81 Dual Determination on Extracellular Vesicles from Breast Cancer Patients.

This study is focused on identifying novel epithelial markers in circulating extracellular vesicles (EVs) through the development of a dual sandwich-type electrochemical paper-based immunosensor for Claudin 7 and CD81 determination, as well as its validation in breast cancer (BC) patients. This immunosensor allows for rapid, sensitive, and label-free detection of these two relevant BC biomarkers. Under optimum conditions, the limit of detection for Claudin 7 was 0.4 pg mL-1, with a wide linear range of 2 to 1000 pg mL-1, while for CD81, the limit of detection was 3 pg mL-1, with a wide linear range of 0.01 to 10 ng mL-1. Finally, we validated Claudin 7 and CD81 determination in EVs from 60 BC patients and 20 healthy volunteers, reporting higher diagnostic accuracy than the one observed with classical diagnostic markers. This analysis provides a low-cost, specific, versatile, and user-friendly strategy as a robust and reliable tool for early BC diagnosis.

Prognostic factors in patients with vulvar cancer: the VULCAN study.

OBJECTIVE: This study aimed to analyze the prognostic factors for overall and progression-free survival in patients with vulvar cancer. METHODS: This international, multicenter, retrospective study included 2453 patients diagnosed with vulvar cancer at 100 different institutions. Inclusion criteria were institutional review board approval from each collaborating center, pathologic diagnosis of invasive carcinoma of the vulva, and primary treatment performed at the participating center. Patients with intraepithelial neoplasia or primary treatment at non-participating centers were excluded. Global survival analysis and squamous cell histology subanalysis was performed. RESULTS: After excluding patients due to incomplete data entry, 1727 patients treated for vulvar cancer between January 2001 and December 2005 were registered for analysis (1535 squamous, 42 melanomas, 38 Paget's disease and 112 other histologic types). Melanomas had the worse prognosis (p=0.02). In squamous vulvar tumors, independent factors for increase in local recurrence of vulvar cancer were: no prior radiotherapy (p<0.001) or chemotherapy (p=0.006), and for distant recurrence were the number of positive inguinal nodes (p=0.025), and not having undergone lymphadenectomy (p=0.03) or radiotherapy (p<0.001), with a HR of 1.1 (95% CI 1.2 to 1.21), 2.9 (95% CI 1.4 to 6.1), and 3.1 (95% CI 1.7 to 5.7), respectively. Number of positive nodes (p=0.008), FIGO stage (p<0.001), adjuvant chemotherapy (p=0.001), tumor resection margins (p=0.045), and stromal invasion >5 mm (p=0.001) were correlated with poor overall survival, and large case volume (≥9 vs <9 cases per year) correlated with more favorable overall survival (p=0.05). CONCLUSIONS: Advanced patient age, number of positive inguinal lymph nodes, and lack of adjuvant treatment are significantly associated with a higher risk of relapse in patients with squamous cell vulvar cancer. Case volume per treating institution, FIGO stage, and stromal invasion appear to impact overall survival significantly. Future prospective trials are warranted to establish these prognostic factors for vulvar cancer.

EGFR detection in extracellular vesicles of breast cancer patients through immunosensor based on silica-chitosan nanoplatform.

In the present work, we designed a microfluidic electrochemical immunosensor with enough sensibility and precision to quantify epithermal growth factor receptor (EGFR) in plasma extracellular vesicles (EVs) of plasma from breast cancer patients. The sensor employs SiNPs coated with chitosan (SiNPs-CH) as reaction's platform, based on the covalently immobilization of monoclonal anti-EGFR on SiNPs-CH retained in the central channel (CC) of the microfluidic device. The synthetized SiNPs-CH were characterized by UV-visible spectroscopy (UV-visible), energy dispersive spectrometry (EDS), Nanoparticle Tracking Analysis (NTA) and transmission electron microscopy (TEM). EGFR was quantified by a direct sandwich immunoassay measuring through a horseradish peroxidase (HRP)-conjugated anti-EGFR. The enzymatic product (benzoquinone) was detected by reduction at - 100 mV on a sputtering gold electrode. The measured current was directly proportional to the level of EGFR in human serum samples. The linear range was from 0 ng mL-1 to 50 ng mL-1. The detection limit was 1.37 pg mL-1, and the within- and between-assay coefficients of variation were below 6.25%. Finally, plasma samples from 30 early breast cancer patients and 20 healthy donor were analyzed by the novel method. EGFR levels in EVs (EVs-EGFR) were significantly higher than in the healthy control group (p = 0.002) and also, more sensitivity and specificity than normal serum markers like CEA and CA15.3 has been observed. EVs-EGFR concentration correlates with EGFR tumor status (p = 0.0003) as well as it correlate with the tumor size and pathological grade. To conclude, plasma EVs are suitable for proteomic characterization of cancer disease, as long as the employed method has sufficient sensitivity, like the case of immune-electrochemical nanosensors with incremented reaction surface.

Negative Social Evaluation Impairs Executive Functions in Adolescents With Excess Weight: Associations With Autonomic Responses.

BACKGROUND: Adolescents with excess weight suffer social stress more frequently than their peers with normal weight. PURPOSE: To examine the impact of social stress, specifically negative social evaluation, on executive functions in adolescents with excess weight. We also examined associations between subjective stress, autonomic reactivity, and executive functioning. METHODS: Sixty adolescents (aged 13-18 years) classified into excess weight or normal weight groups participated. We assessed executive functioning (working memory, inhibition, and shifting) and subjective stress levels before and after the Trier Social Stress Task (TSST). The TSST was divided into two phases according to the feedback of the audience: positive and negative social evaluation. Heart rate and skin conductance were recorded. RESULTS: Adolescents with excess weight showed poorer executive functioning after exposure to TSST compared with adolescents with normal weight. Subjective stress and autonomic reactivity were also greater in adolescents with excess weight than adolescents with normal weight. Negative social evaluation was associated with worse executive functioning and increased autonomic reactivity in adolescents with excess weight. CONCLUSIONS: The findings suggest that adolescents with excess weight are more sensitive to social stress triggered by negative evaluations. Social stress elicited deterioration of executive functioning in adolescents with excess weight. Evoked increases in subjective stress and autonomic responses predicted decreased executive function. Deficits in executive skills could reduce cognitive control abilities and lead to overeating in adolescents with excess weight. Strategies to cope with social stress to prevent executive deficits could be useful to prevent future obesity in this population.

Increased food choice-evoked brain activation in adolescents with excess weight: Relationship with subjective craving and behavior.

OBJECTIVE: We used functional magnetic resonance imaging (fMRI) to assess brain regions associated with food choices between appetizing (i.e., high sugar, high fat) and plain food in adolescents with excess weight and those with normal weight. The associations between choice-evoked brain activation and subjective food craving and behavioral food choices were also evaluated. METHODS: Seventy-three adolescents (aged 14-19 years), classified into excess weight (n = 38) or normal weight (n = 39) groups, participated in the study. We used a food-choice fMRI task, between appetizing and plain food, to analyse brain activation differences between groups. Afterwards, participants assessed their "craving" for each food presented in the scanner. RESULTS: Adolescents with excess weight showed higher brain activation in frontal, striatal, insular and mid-temporal regions during choices between appetizing and standard food cues. This pattern of activations correlated with behavioral food choices and subjective measures of craving. CONCLUSIONS: Our findings suggest that adolescents with excess weight have greater food choice-related brain reactivity in reward-related regions involved in motivational and emotional responses to food. Increased activation in these regions is generally associated with craving, and increased dorsolateral prefrontal cortex is specifically associated with appetizing food choices among adolescents with excess weight, which may suggest greater conflict in these decisions. These overweight- and craving-associated patterns of brain activation may be relevant to decision-making about food consumption.

A comprehensive study of circulating tumour cells at the moment of prostate cancer diagnosis: biological and clinical implications of EGFR, AR and SNPs.

Circulating tumor cells (CTCs) have been recently accepted as prognostic markers in metastatic prostate cancer (PCa). However, very few studies have analyzed their role in early-stage PCa. The aim of this research is to study the value of CTCs at the moment of PCa diagnosis and to identify different subpopulations of CTCs. Patients with PSA value > 4 ng/ml and clinical suspicion of PCa were included. Samples were collected immediately before prostatic biopsy. CTCs were isolated by immunomagnetic technique using a multi-CK specific antibody. Molecular expression of EGFR and AR in the tissue was analysed by real-time PCR. Up to eight different SNPs in patients' blood DNA were studied. In a total of 86 patients, the CTC detection rate was 18.6%. The sensitivity, specificity, positive and negative predictive values of CTCs to detect PCa was 14.2%, 78.4%, 31.2% and 57.4%, respectively. Up to 75% of CTC-positive patients were AR-negative. A direct association was found between the expression of AR in the prostatic tissue and the presence of CTCs in blood (p<0.05). We observed an inverse relation between the expression of EGFR in the tissue and the expression of AR in the CTCs. No significant association between SNPs and CTCs was found. The low detection rate of CTCs in early-stage PCa limits their role as a diagnostic marker. Nevertheless, we show that they may hide important prognostic information. Overexpression of AR in the prostate may facilitate cell dissemination.

Increased corticolimbic connectivity in cocaine dependence versus pathological gambling is associated with drug severity and emotion-related impulsivity.

Neural biomarkers for the active detrimental effects of cocaine dependence (CD) are lacking. Direct comparisons of brain connectivity in cocaine-targeted networks between CD and behavioural addictions (i.e. pathological gambling, PG) may be informative. This study therefore contrasted the resting-state functional connectivity networks of 20 individuals with CD, 19 individuals with PG and 21 healthy individuals (controls). Study groups were assessed to rule out psychiatric co-morbidities (except alcohol abuse and nicotine dependence) and current substance use or gambling (except PG). We first examined global connectivity differences in the corticolimbic reward network and then utilized seed-based analyses to characterize the connectivity of regions displaying between-group differences. We examined the relationships between seed-based connectivity and trait impulsivity and cocaine severity. CD compared with PG displayed increased global functional connectivity in a large-scale ventral corticostriatal network involving the orbitofrontal cortex, caudate, thalamus and amygdala. Seed-based analyses showed that CD compared with PG exhibited enhanced connectivity between the orbitofrontal and subgenual cingulate cortices and between caudate and lateral prefrontal cortex, which are involved in representing the value of decision-making feedback. CD and PG compared with controls showed overlapping connectivity changes between the orbitofrontal and dorsomedial prefrontal cortices and between amygdala and insula, which are involved in stimulus-outcome learning. Orbitofrontal-subgenual cingulate cortical connectivity correlated with impulsivity and caudate/amygdala connectivity correlated with cocaine severity. We conclude that CD is linked to enhanced connectivity in a large-scale ventral corticostriatal-amygdala network that is relevant to decision making and likely to reflect an active cocaine detrimental effect.

Dormant Circulating Tumor Cells in Prostate Cancer: Therapeutic, Clinical and Biological Implications.

Circulating Tumor Cells (CTCs) are a valuable prognostic factor in several solid tumors. By understanding the biological characteristics of CTCs we could better understand the biology of metastasis. CTCs usually adopt a dormant state that is believed to be a strategy to survive in extreme conditions. To enter a dormant state, CTCs undergo numerous phenotypic, genetic and functional mutations that significantly affect the efficacy of the therapies used to kill dormant CTCs. Hence, understanding the biological events involved in the dormancy process of CTCs would allow the identification of new therapeutic targets. Some experimental studies or preclinical models have explored these biological events, as well as the molecular factors that contribute to the maintenance of and release from dormancy. However, few studies have assessed the effects of anticancer therapies on dormant cells. This study reviews current the data currently available on cell dormancy mechanisms in prostate cancer, with a special focus on the functional, genetic and phenotypic plasticity of CTCs and their potential implications in the clinical and therapeutic management of prostate cancer.

Cocaine-specific neuroplasticity in the ventral striatum network is linked to delay discounting and drug relapse.

AIMS: To contrast functional connectivity on ventral and dorsal striatum networks in cocaine dependence relative to pathological gambling, via a resting-state functional connectivity approach; and to determine the association between cocaine dependence-related neuroadaptations indexed by functional connectivity and impulsivity, compulsivity and drug relapse. DESIGN: Cross-sectional study of 20 individuals with cocaine dependence (CD), 19 individuals with pathological gambling (PG) and 21 healthy controls (HC), and a prospective cohort study of 20 CD followed-up for 12 weeks to measure drug relapse. SETTING AND PARTICIPANTS: CD and PG were recruited through consecutive admissions to a public clinic specialized in substance addiction treatment (Centro Provincial de Drogodependencias) and a public clinic specialized in gambling treatment (AGRAJER), respectively; HC were recruited through community advertisement in the same area in Granada (Spain). MEASUREMENTS: Seed-based functional connectivity in the ventral striatum (ventral caudate and ventral putamen) and dorsal striatum (dorsal caudate and dorsal putamen), the Kirby delay-discounting questionnaire, the reversal-learning task and a dichotomous measure of cocaine relapse indicated with self-report and urine tests. FINDINGS: CD relative to PG exhibit enhanced connectivity between the ventral caudate seed and subgenual anterior cingulate cortex, the ventral putamen seed and dorsomedial pre-frontal cortex and the dorsal putamen seed and insula (P≤0.001, kE=108). Connectivity between the ventral caudate seed and subgenual anterior cingulate cortex is associated with steeper delay discounting (P≤0.001, kE=108) and cocaine relapse (P≤0.005, kE=34). CONCLUSIONS: Cocaine dependence-related neuroadaptations in the ventral striatum of the brain network are associated with increased impulsivity and higher rate of cocaine relapse.

Study of antitumor activity in breast cell lines using silver nanoparticles produced by yeast.

In the present article, we describe a study of antitumor activity in breast cell lines using silver nanoparticles (Ag NPs) synthesized by a microbiological method. These Ag NPs were tested for their antitumor activity against MCF7 and T47D cancer cells and MCF10-A normal breast cell line. We analyzed cell viability, apoptosis induction, and endocytosis activity of those cell lines and we observed that the effects of the biosynthesized Ag NPs were directly related with the endocytosis activity. Moreover, Ag NPs had higher inhibition efficacy in tumor lines than in normal lines of breast cells, which is due to the higher endocytic activity of tumor cells compared to normal cells. In this way, we demonstrate that biosynthesized Ag NPs can be an alternative for the treatment of tumors.

Social stress increases cortisol and hampers attention in adolescents with excess weight.

OBJECTIVE: To experimentally examine if adolescents with excess weight are more sensitive to social stress and hence more sensitive to harmful effects of stress in cognition. DESIGN AND METHODS: We conducted an experimental study in 84 adolescents aged 12 to 18 years old classified in two groups based on age adjusted Body Mass Index percentile: Normal weight (n=42) and Excess weight (n=42). Both groups were exposed to social stress as induced by the virtual reality version of the Trier Social Stress Task--participants were requested to give a public speech about positive and negative aspects of their personalities in front of a virtual audience. The outcome measures were salivary cortisol levels and performance in cognitive tests before and after the social stressor. Cognitive tests included the CANTAB Rapid Visual Processing Test (measuring attention response latency and discriminability) and the Iowa Gambling Task (measuring decision-making). RESULTS: Adolescents with excess weight compared to healthy weight controls displayed increased cortisol response and less improvement of attentional performance after the social stressor. Decision-making performance decreased after the social stressor in both groups. CONCLUSION: Adolescents who are overweight or obese have increased sensitivity to social stress, which detrimentally impacts attentional skills.

Ephrin reverse signaling mediates palatal fusion and epithelial-to-mesenchymal transition independently of Tgfß3.

The mammalian secondary palate forms from shelves of epithelia-covered mesenchyme that meet at midline and fuse. The midline epithelial seam (MES) is thought to degrade by apoptosis, epithelial-to-mesenchymal transition (EMT), or both. Failure to degrade the MES blocks fusion and causes cleft palate. It was previously thought that transforming growth factor ß3 (Tgfß3) is required to initiate fusion. Members of the Eph tyrosine kinase receptor family and their membrane-bound ephrin ligands are expressed on the MES. We demonstrated that treatment of mouse palates with recombinant EphB2/Fc to activate ephrin reverse signaling (where the ephrin acts as a receptor and transduces signals from its cytodomain) was sufficient to cause mouse palatal fusion when Tgfß3 signaling was blocked by an antibody against Tgfß3 or by an inhibitor of the TgfßrI serine/threonine receptor kinase. Cultured palatal epithelial cells traded their expression of epithelial cell markers for that of mesenchymal cells and became motile after treatment with EphB2/Fc. They concurrently increased their expression of the EMT-associated transcription factors Snail, Sip1, and Twist1. EphB2/Fc did not cause apoptosis in these cells. These data reveal that ephrin reverse signaling directs palatal fusion in mammals through a mechanism that involves EMT but not apoptosis and activates a gene expression program not previously associated with ephrin reverse signaling.

miRNA in situ hybridization in circulating tumor cells--MishCTC.

Circulating tumor cells (CTCs) must be phenotypically and genetically characterized before they can be utilized in clinical applications. Here, we present the first protocol for the detection of miRNAs in CTCs using in situ hybridization (ISH) combined with immunomagnetic selection based on cytokeratin (CK) expression and immunocytochemistry. Locked-Nucleic Acid (LNA) probes associated with an enzyme-labeled fluorescence (ELF) signal amplification approach were used to detect miRNA-21 in CTCs. This protocol was optimized using both epithelial tumor (MDA-MB468) and epithelial non-tumor (MCF-10A) cell lines, and miRNA-21 was selected as the target miRNA because of its known role as an onco-miRNA. Hematopoietic cells do not express miRNA-21; thus, miRNA-21 is an ideal marker for detecting CTCs. Peripheral blood samples were taken from 25 cancer patients and these samples were analyzed using our developed protocol. Of the 25 samples, 11 contained CTCs. For all 11 CTC-positive samples, the isolated CTCs expressed both CK and miRNA-21. Finally, the protocol was applied to monitor miRNA-21 expression in epithelial to mesenchymal transition (EMT)-induced MCF-7 cells, an epithelial tumor cell line. CK expression was lost in these cells, whereas miRNA-21 was still expressed, suggesting that miRNA-21 might be a good marker for detecting CTCs with an EMT phenotype.

EMT and EGFR in CTCs cytokeratin negative non-metastatic breast cancer.

Circulating tumor cells (CTCs) are frequently associated with epithelial-mesenchymal transition (EMT).The objective of this study was to detect EMT phenotype through Vimentin (VIM) and Slug expression in cytokeratin (CK)-negative CTCs in non-metastatic breast cancer patients and to determine the importance of EGFR in the EMT phenomenon. In CK-negative CTCs samples, both VIM and Slug markers were co-expressed in the most of patients. Among patients EGFR+, half of them were positive for these EMT markers. Furthermore, after a systemic treatment 68% of patients switched from CK- to CK+ CTCs. In our experimental model we found that activation of EGFR signaling by its ligand on MCF-7 cells is sufficient to increase EMT phenotypes, to inhibit apoptotic events and to induce the loss of CK expression. The simultaneous detection of both EGFR and EMT markers in CTCs may improve prognostic or predictive information in patients with operable breast cancer.

Therapeutic adherence in primary care depressed patients: a longitudinal study.

INTRODUCTION: Lack of adherence has been associated to lower efficacy of anti-depressant treatment, increasing the risk of recurrence and persistence of clinical symptoms. Patients with poor medication adherence have more concomitant medical illnesses and somatic symptoms. Furthermore, this increases use of healthcare services. METHOD: Longitudinal and observational study on therapeutic adherence level in depressive outpatients treated in 3 Primary Care (PC) centers. Eight evaluations during 6 months were carried out in 29 patients over 18, with DSM-IV-TR major depression diagnosis. The purpose of the present study was to determine adherence level, to analyze socio-demographic factors and clinical profiles involved in adherence, and to observe the evolution of depressive symptoms. RESULTS: Good therapeutic adherence was observed in 72.4% of patients. Significant differences in the Drug Attitude Inventory (U=107.5; p=0.036) were found. This tool evaluates the perceived effect of the medication, with a better perception observed in adherent patients. In those patients a progressive reduction on the Hamilton Depression Scale was found over the course of six monthly follow-up visits, with clinical remission observed in month 4. The analysis of survival rate did not reveal any significant difference between the two groups [Log Rank (χ2=1.610, p=0.205)]. CONCLUSIONS: The therapeutic adherence observed in this longitudinal PC study is high, and it is associated with an improvement in the illness. A better perceived effect of the treatment showed a significant connection to an improvement in symptoms of depression.

Roles of notch signalling in mandibular condylar cartilage.

IMPORTANCE: Notch proteins are cell surface transmembrane spanning receptors which mediate critically important cellular functions through direct cell-cell contact. Interactions between Notch receptors and their ligands regulate cell fate decisions such differentiation, proliferation and apoptosis in numerous tissues. We have previously shown using immunohistochemistry that Notch1 is localized primarily to the prechondroblastic (chondroprogenitor) layer of the mandibular condylar cartilage (MCC). OBJECTIVE: To test if Notch signalling changes patterns of proliferation and differentiation in the MCC and to investigate if Notch signalling acts downstream of Fibroblast Growth Factor 2 (FGF-2). METHODS: Condylar cartilage explants were cultured over serum-free DMEM containing either 0 or 50nM DAPT, a Notch signal inhibitor. Explants were used for RNA extraction and immunohistochemistry. RESULTS: Analysis of gene array data demonstrated that the perichondrial layer of the MCC is rich in Notch receptors (Notch 3 and 4) and Notch ligands (Jagged and Delta) as well as various downstream facilitators of Notch signalling. Disruption of Notch signalling in MCC explants decreased proliferation (Cyclin B1 expression) and increased chondrocyte differentiation (Sox9 expression). Moreover, we found that the actions of FGF-2 in MCC are mediated in part by Notch signalling. CONCLUSION: These data suggest that Notch signalling contributes to the regulation of proliferation and differentiation in the MCC.

Cell-matrix and cell-cell interactions of human gingival fibroblasts on three-dimensional nanofibrous gelatin scaffolds.

An in-depth understanding of the interactions between cells and three-dimensional (3D) matrices (scaffolds) is pivotal to the development of novel biomaterials for tissue regeneration. However, it remains a challenge to find suitable biomimetic substrates and tools to observe cell-material and cell-cell interactions on 3D matrices. In the present study, we developed biomimetic nanofibrous 3D gelatin scaffolds (3D-NF-GS) and utilized confocal microscopy combined with a quantitative analysis approach to explore cell-matrix and cell-cell interactions on the 3D-NF-GS. Human gingival fibroblasts (HGFs) migrated throughout the 3D-NF-GS by 5 days and formed stable focal adhesions by 14 days. The focal adhesions were detected using integrin-ß1, phospho-paxillin and vinculin expression, which were quantified from specific wavelength photon data generated using a spectral separation confocal microscope. As the cells became more confluent after 14 days of culture, cell-cell communication via gap junctions increased significantly. Collagen I matrix production by HGFs on 3D-NF-GS was visualized and quantified using a novel approach incorporating TRITC label in the scaffolds. Based on confocal microscopy, this study has developed qualitative and quantitative methods to study cell-matrix and cell-cell interactions on biomimetic 3D matrices, which provides valuable insights for the development of appropriate scaffolds for tissue regeneration.