Effects of tobacco products on the attachment and growth of periodontal ligament fibroblasts

BACKGROUND: Cigarette smoking is one of the most significant risk factors in the development and further advancement of inflammatory periodontal disease, however, the role of either nicotine or its primary metabolite cotinine in the progression of periodontitis is unclear. This study aimed to investigate the effects of nicotine and cotinine on the attachment and growth of fibroblasts derived from human periodontal ligament (PDL).

METHODS: Primary cultures were prepared from the roots of extracted premolar teeth. Cells were used at both low (P3 to P5) and high (P11 to P13) passage. Cell numbers were determined over 14 days using either the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay or with a Coulter counter. Cultures were exposed to culture medium supplemented with 1) 15% fetal calf serum (FCS) only; 2) 1% FCS only; 3) 1% FCS and nicotine (concentration range 5 ng/ml to 10 mg/ml); or 4) 1% FCS and cotinine (concentration range 0.5 ng/ml to 10 microg/ml).

RESULTS: Nicotine significantly (P <0.05, by ANOVA) inhibits attachment and growth of low passage cells at concentrations >1 mg/ml and high passage PDL fibroblasts at concentrations >0.5 mg/ml. Cotinine, at the highest concentration used (10 microg/ml), appeared to inhibit attachment and growth of both low and high passage fibroblasts but this was not statistically significant (P >0.05, by ANOVA).

CONCLUSIONS: Tobacco products inhibit attachment and growth of human PDL fibroblasts. This may partly explain the role of these substances in the progression of periodontitis.

Neuropeptide Y in GCF in periodontal health and disease

Objectives: To determine whether neuropeptide Y (NPY) is present in gingival crevicular fluid (GCF) in both periodontal health and disease and to study the relationship of NPY with periodontal inflammation. Methods: GCF samples (30 s) were collected from one site with both pocket depth (>4mm) and loss of periodontal attachment (>4mm) in 20 patients with chronic periodontitis (mean age 41.4, SD 9.6 yrs; 10 m, 10 f). GCF was also collected from clinically healthy sites (< 3mm, no bleeding on probing) in 20 subjects with no periodontitis (mean age 37.4, SD 11.7; 10 m, 10 f). GCF was collected using the periopaper strip method, diluted in 500 ul of phosphate-buffered saline and stored at –70°C. Samples were analysed in duplicate for NPY by radioimmunoassay. NPY levels were compared using the Mann-Whitney test. Results: Measurable NPY was present in all the GCF samples collected from healthy subjects. NPY was below the level of detection in 4 (20%) of the diseased subjects. There was considerable variability in the amount of NPY collected from both groups. There were no differences between the levels of NPY measured in males compared with females in either the healthy or diseased groups. Significantly more (P< 0.0001) NPY (pg) was collected from healthy subjects (Median 165, IQR 80; mean 161, SD 64) than diseased subjects (Median 37.5, IQR 56.3; mean 39.8, SD 35.1). There was more variability in the NPY concentration (pg/ul) which was also significantly higher in healthy (Median 575.7, IQR 562.3; mean 645.7, SD 416.7) compared with diseased subjects (Median 43.6, IQR 117.4; mean 96.4, SD 124.5). Conclusions: It is concluded that the levels of NPY in GCF sampled

The role of LL-37 in periodontal wound healing

Introduction: As a result of chronic inflammation during periodontal disease the junctional epithelium becomes micro-ulcerated. The inflammatory process is mediated by both bacterial and host cell products. Host defence peptides such as defensins, secretory leucocyte protease inhibitor (SLPI) and the sole human cathelicidin, LL-37, are secreted by both periodontal cells and neutrophils into gingival crevicular fluid (GCF). They have the ability to modulate the immune response in periodontitis and are thought to have a potential role in periodontal wound healing. Objectives: The aims of this study were to determine the role of LL-37 in the production of Interleukin (IL)-8, IL-6, hepatocyte growth factor (HGF) and basic-fibroblast growth factor (bFGF) by gingival fibroblasts. The role of LL-37 in modulating total matrix metalloproteinase (MMP) activity and expression of tissue inhibitors of metalloproteinase (TIMP)-1 and -2 by gingival fibroblasts was also investigated. Methods: Primary gingival fibroblasts were co-cultured with concentrations of LL-37 (1, 5 and 10µg/ml) for 24 hours and their supernatants tested for levels of IL-8 and IL-6, HGF, bFGF, TIMP-1 and TIMP-2 by ELISA. Rates of MMP turnover in the supernatants were tested by fluorogenic assay using fluorescence resonance energy transfer (FRET) peptide substrates. Cytotoxicity was measured by MTT assay. Statistical significance was measured using the independent t-test and p<0.05 was considered significant. Results: LL-37 significantly upregulated levels of IL-8, IL-6, HGF, bFGF and TIMP-1 (p<0.05) in a dose-dependent fashion. LL-37 significantly decreased the total MMP activity (p<0.05). None of the LL-37 concentrations tested were cytotoxic to gingival fibroblasts. Conclusion: These results indicate that LL-37 is involved in periodontal wound healing. LL-37 increased levels of proinflammatory cytokines and increased levels of growth factors involved in re-epithelialisation. LL-37 has the ability to regulate remodelling of the periodontium by controlling MMP overactivity both directly and by stimulating production of inhibitors by gingival fibroblasts.

Eppin: A Novel Protein Possessing Antimicrobial Properties Against Oral Pathogens

Background: Epididymal protease inhibitor (eppin) is a dual motif protein belonging to the whey acidic protein (WAP) family. Although expressed in numerous different tissues, to date, its functional characterisation is limited. It has been shown to exhibit antibacterial activity against Gram-negative bacteria (Escherichia coli) and antiprotease activity against some proteases of the serine protease family. We are interested in determining the role of eppin in innate immune defence. Objectives: This study aims to determine eppin's potential function in the innate immune response in the oral cavity by investigating the antimicrobial activity of eppin against relevant oral pathogens. Methods: Eppin was recombinantly expressed in E. coli cells and purified by immobilised metal affinity chromatography (IMAC). The antimicrobial effects of the protein were then assessed against two oral pathogens, Fusobacterium nucleatum and Candida albicans, using a double layer radial diffusion assay. Results: Eppin displayed antimicrobial activities against both oral pathogens tested and these activities were shown to be comparable to the well characterised antimicrobial peptide, LL-37. The antifungal effects of eppin were shown to be more potent than those of the human cathelicidin, LL-37. Conclusions: Eppin has been shown to possess both antibacterial and antifungal properties against oral pathogens, suggesting an important role for this protein in the innate immune response in the oral cavity. This study furthers our knowledge of the physiological role exerted by eppin and its possible role in the modulation of chronic diseases such as periodontitis and oral candidiasis.

MMP-8 Activity Profiling In GCF Samples

Background: In healthy tissues a family of enzymes known as matrix metalloproteinases (MMPs) play an important role in regulating turnover and metabolism of connective tissue collagen. MMPs have been implicated in a wide variety of pathological conditions including periodontal disease. MMP-8 has been extensively studied in periodontal health and disease using enzyme-linked immunosorbent assay (ELISA). Although ELISA quantifies the presence of the MMP-8 protein, it is not possible to determine enzyme activity using this method. Furthermore, since members of the MMP family have poor substrate sequence specificity, a peptide substrate alone cannot differentiate the activity of MMP-8 from other MMPs that may be present in biological samples. Objectives: In the present study, a method to specifically measure MMP-8 activity in gingival crevicular fluid (GCF) samples was developed. Methods: GCF was collected from healthy patients and those with periodontal disease using Perio paper strips. Samples were stored frozen until required for analysis. A specific MMP-8 antibody was used to coat 96 well microtitre plates to selectively remove MMP-8 from the GCF samples. Following a washing step, the activity of bound MMP-8 was measured over 70 minutes using a fluorogenic (FRET) substrate. Results: GCF from healthy subjects exhibited basal MMP-8 activity but in diseased samples MMP-8 activity was significantly higher. Minimal binding of other recombinant MMPs to the specific MMP-8 antibody was observed in cross-reactivity studies. Conclusion: We show for the first time that MMP-8 activity was significantly increased in GCF from periodontitis sites compared with activity levels in healthy sites. Further studies of MMP-8 activity in GCF samples should improve our understanding of its destructive role in periodontal disease.

Design, synthesis and validation of an inhibitor of CGRP degradation

Introduction: In addition to their afferent role in detection and signalling noxious stimuli, neuropeptide-containing sensory nerves may initiate and maintain chronic inflammation in diseases such as periodontitis by an efferent process known as neurogenic inflammation. Neuropeptides are susceptible to cleavage by peptidases, and therefore, the exact location and level of expression of peptidases are major determinants of neuropeptide action. Previous studies in our laboratory showed that enzyme components of gingival crevicular fluid (GCF) from periodontitis sites selectively inactivated the neuropeptide calcitonin gene-related peptide (CGRP), known to have a role in inhibiting osteoclastic bone resorption. Objectives: The aim of this study was to design and synthesise a specific inhibitor to prevent the degradation of CGRP by components of GCF. Methods: A hydroxamate-based inhibitor with a biotinylated tag was designed to ensure selectivity for CGRP and ease of use for future purification strategies. The biotinylated peptide hydroxamate contained the P1-P4 amino acid sequence of the potential CGRP cleavage site and was synthesised by solid-phase methods using standard Fmoc chemistry. Inhibition of CGRP metabolism by GCF was determined by MALDI-mass spectrometry (MALDI-MS) using pooled GCF samples from periodontitis patients as a crude source of the CGRP-degrading enzyme. Results: MALDI-MS analysis of CGRP degradation showed almost complete inhibition in the presence of the biotinylated inhibitor. Our results showed that the rate-limiting step in the cleavage of CGRP is endopeptidase cleavage, followed by carboxypeptidase attack. Conclusion: This study demonstrates that the enzyme component of GCF responsible for the degradation of CGRP can be inhibited by a biotinylated hydroxamate modelled on a potential endopeptidase cleavage site. The biotin tag on the inhibitor will facilitate our future purification of the CGRP-cleavage enzyme using a streptavidin-agarose column.

LL-37 Degradation by GCF: A Role for Porphyromonas gingivalis Proteinases?

Background: LL-37, composed of 37 amino acid residues, is an innate host defence peptide of the cathelicidin family. It is expressed by neutrophils, monocytes and epithelial cells and exhibits both anti-bacterial and immunomodulatory properties. LL-37 is however prone to proteolytic degradation by proteinases, thus potentially limiting its inherent host defence properties in the inflammatory milieu. Objectives: The present study was designed to determine whether LL-37 was degraded by components of gingival crevicular fluid (GCF) from healthy subjects or those with periodontitis. In addition, we aimed to deduce whether degradation of the peptide was accelerated in GCF samples which were determined to be positive for the periodontopathic bacterium Porphyromonas gingivalis. Methods: GCF and bacterial plaque samples, pre- and post non-surgical periodontal treatment, were collected from 4 individual sites in patients presenting with advanced periodontitis. In healthy subjects, GCF samples only were collected. Plaque samples were analysed by QPCR for the presence or absence of P. gingivalis. Pooled GCF samples from healthy sites; periodontitis sites which were P. gingivalis negative (Pg-); or periodontitis sites which were P. gingivalis positive (Pg+), were incubated with synthetic LL-37 for 0 – 180 min. The degradation products were then analysed by matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS). Results: LL-37 was not degraded when incubated with GCF from healthy subjects. In contrast, LL-37 was degraded after 30 min when incubated with Pg- GCF. However degradation of LL-37 was apparent after only 2 min incubation with Pg+ GCF and the parent molecule was almost completely degraded after 30 min. Conclusions: The rapid degradation of LL-37, particularly in Pg+ sites, highlights the limited role which this host defence peptide may play in the presence of biologically active proteinases. It also underscores a potent virulence mechanism of P. gingivalis used to circumvent innate host responses.

Immunocytochemical Localization of Substance P Receptors (NK-1 Receptors) in Human Gingival Tissue

Substance P (SP) is a member of the structurally related family of neuropeptides known as the tachykinins. In addition to neurotransmitter roles, the tachykinins are also known to modulate local inflammation which depends on signalling between the neuropeptide molecules and target cells and tissues. SP mediates its effects through a specific receptor, known as the substance P receptor or the neurokinin 1 (NK-1) receptor. The NK-1 receptor is a G-protein associated integral membrane protein and although it has been studied in a wide range of tissues, to date there has been no published data on the localisation of the NK-1 receptor in human gingival tissue. Objective: The aim of this study was to examine the distribution of the NK-1 receptor in human gingival tissue using immunocytochemistry. Method: Gingival tissue was obtained from patients undergoing periodontal surgery. Tissue was fixed in paraformaldehyde and embedded in wax for sectioning. Sections were dewaxed in xylene and then rehydrated in alcohols and phosphate buffered saline. Rehydrated sections were probed with rabbit polyclonal antibody to human NK-1 receptor which was subsequently detected using anti-rabbit horseradish peroxidase conjugate and diaminobenzidine as substrate. Results: Immunocytochemistry revealed that the NK-1 receptor was distributed along nerve fibres and blood vessel endothelial cells, suggesting these areas are main targets for the actions of SP via the NK-1 receptor. Conclusion: This is the first immunocytochemical report of NK-1 receptors in human gingival tissue and provides evidence for possible NK-1 mediated biological effects of SP in human gingival tissue from periodontitis patients.

CGRP-cleavage enzyme detection using a biotinylated hydroxymate affinity probe

Introduction: Neuropeptides contribute to the pathophysiology of peripheral inflammation and a neurogenic component has been described for many inflammatory diseases, including periodontitis. Neuropeptides are susceptible to cleavage by peptidases and therefore the exact location and level of expression of peptidases are major determinants of neuropeptide action. Previous studies by our research group suggested that levels of the neuropeptide calcitonin gene-related peptide (CGRP) may be regulated by peptidases present in gingival crevicular fluid (GCF). Objectives: The aim of this work was to purify and partially characterize the GCF enzyme responsible for CGRP degradation using a biotinylated hydroxymate affinity probe (based on the P1-P4 amino acid sequence of the observed cleavage site) which we previously showed to inhibit CGRP degradation. Methods: Pooled healthy and pooled periodontitis GCF samples were subject to a pre-clear step with magnetic streptavadin beads. Healthy and diseased samples were incubated with the biotinylated hydroxymate probe (20 uM) after which biotinylated proteins were purified from the sample using magnetic streptavadin beads. Bound proteins were subjected to SDS-PAGE and western blotting. Biotin incorporated proteins were disclosed using a streptavadin horse radish peroxidase conjugate. Results: A band was disclosed in the periodontitis pooled sample at a molecular weight of approximately 60 kDa. The band was absent in the pooled healthy samples. As expected, when periodontitis samples were pre-boiled to denature proteins before the addition of the hydroxymate probe, no biotin incorporated band was present. Conclusions: This work demonstrates the purification and disclosure of a protein found specifically in periodontitis which binds to the specific biotinylated hydroxymate affinity probe based on the cleavage site of CGRP only when in its native form. We intend to scale up the sample size thus allowing the identification of the putative CGRP degrading peptidase using MALDI-mass spectrometry.
Funded by an IADR/GlaxoSmithKline Innovation in Oral Care Award

Lateral bias and temperament in the domestic cat (Felis silvestris)

Research points to a relationship between lateralization and emotional functioning in humans and many species of animal. The present study explored the association between paw preferences and emotional functioning, specifically temperament, in a species thus far overlooked in this area, the domestic cat. Thirty left-pawed, 30 right-pawed, and 30 ambilateral pet cats were recruited following an assessment of their paw preferences using a food-reaching challenge. The animals’ temperament was subsequently assessed using the Feline Temperament Profile (FTP). Cats’ owners also completed a purpose-designed cat temperament (CAT) scale. Analysis revealed a significant relationship between lateral bias and FTP and CAT scale scores. Ambilateral cats had lower positive (FTP+) scores, and were perceived as less affectionate, obedient, friendly, and more aggressive, than left or right-pawed animals. Left and right pawed cats differed significantly on 1 trait on the CAT scale, namely playfulness. The strength of the cats’ paw preferences was related to the animals’ FTP and CAT scores. Cats with a greater strength of paw preference had higher FTP + scores than those with a weaker strength of paw preference. Animals with stronger paw preferences were perceived as more confident, affectionate, active, and friendly than those with weaker paw preferences. Results suggest that motor laterality in the cat is strongly related to temperament and that the presence or absence of lateralization has greater implications for the expression of emotion in this species than the direction of the lateralized bias.

The role of glycans in the development and progression of prostate cancer

Prostate cancer is a unique and heterogeneous disease. Currently, a major unmet clinical need exists to develop biomarkers that enable indolent disease to be distinguished from aggressive disease. The prostate is an abundant secretor of glycoproteins of all types, and alterations in glycans are, therefore, attractive as potential biomarkers and therapeutic targets. Despite progress over the past decade in profiling the genome and proteome, the prostate cancer glycoproteome remains relatively understudied. A wide range of alterations in the glycoproteins on prostate cancer cells can occur, including increased sialylation and fucosylation, increased O-β-N-acetylglucosamine (GlcNAc) conjugation, the emergence of cryptic and high-mannose N-glycans and alterations to proteoglycans. Glycosylation can alter protein function and has a key role in many important biological processes in cancer including cell adhesion, migration, interactions with the cell matrix, immune surveillance, cell signalling and cellular metabolism; altered glycosylation in prostate cancer might modify some, or all of these processes. In the past three years, powerful tools such as glycosylation-specific antibodies and glycosylation gene signatures have been developed, which enable detailed analyses of changes in glycosylation. Thus, emerging data on these often overlooked modifications have the potential to improve risk stratification and therapeutic strategies in patients with prostate cancer.

Network signatures based on gene pair expression ratios improve classification and the analysis of muscle-invasive urothelial cancer

Urothelial cancer (UC) is highly recurrent and can progress from non-invasive (NMIUC) to a more aggressive muscle-invasive (MIUC) subtype that invades the muscle tissue layer of the bladder. We present a proof of principle study that network-based features of gene pairs can be used to improve classifier performance and the functional analysis of urothelial cancer gene expression data. In the first step of our procedure each individual sample of a UC gene expression dataset is inflated by gene pair expression ratios that are defined based on a given network structure. In the second step an elastic net feature selection procedure for network-based signatures is applied to discriminate between NMIUC and MIUC samples. We performed a repeated random subsampling cross validation in three independent datasets. The network signatures were characterized by a functional enrichment analysis and studied for the enrichment of known cancer genes. We observed that the network-based gene signatures from meta collections of proteinprotein interaction (PPI) databases such as CPDB and the PPI databases HPRD and BioGrid improved the classification performance compared to single gene based signatures. The network based signatures that were derived from PPI databases showed a prominent enrichment of cancer genes (e.g., TP53, TRIM27 and HNRNPA2Bl). We provide a novel integrative approach for large-scale gene expression analysis for the identification and development of novel diagnostical targets in bladder cancer. Further, our method allowed to link cancer gene associations to network-based expression signatures that are not observed in gene-based expression signatures.

Comportamentos agressivos entre pares e padrões de vinculação: um estudo com jovens adolescentes

O presente estudo, desenvolvido com 237 jovens de três escolas do distrito de Évora, teve como objetivos caracterizar e analisar os comportamentos relacionados com situações de bullying (vitimação, do agressão e observação); e analisar a existência de eventuais relações entre padrões de vinculação e comportamentos agressivos entre pares. Utilizaram-se dois questionários de auto-relato, nomeadamente o QEVE – Questionário de Exclusão Social e Violência Escolar (Martins, 2003), que mede os comportamentos de agressão, vitimação e observação; e o IVIA – Inventário sobre Vinculação para a Infância e a Adolescência (Carvalho, Soares & Baptista, 2006), que analisa um conjunto de comportamentos e representações da vinculação na infância e na adolescência. Ambos os instrumentos mostraram boas qualidades psicométricas. Os resultados mostraram uma clara prevalência de situações de observação, relativamente às situações de agressão e de vitimação. Em todas estas situações, os comportamentos relacionados com a Exclusão Social e Agressão Verbal são os mais frequentes, comparativamente aos comportamentos relacionados com a Agressão Física. Os rapazes apresentaram médias mais elevadas em todas as escalas; os estudantes com menor percepção de suporte social na escola eram os que evidenciavam mais comportamentos de vitimação. No que se refere aos padrões de vinculação, os resultados mostraram que a vinculação segura parece funcionar como um fator protetor para a vitimação e para a agressividade. A partir dos resultados obtidos, serão analisadas pistas para uma intervenção psicoeducativa.

A qualidade do serviço prestado pelos Serviços Académicos da Escola Superior ABC

A globalização dos mercados transformou a realidade em que vivem as nossas organizações criando a necessidade de, face à concorrência cada vez mais agressiva, lutar por uma posição de destaque no mercado onde se encontram, sendo a “qualidade” cada vez mais o fator de destaque. A qualidade vai muito além de um produto, hoje em dia a qualidade estende-se ao ensino passando pelos serviços. A qualidade preocupa não só a indústria como todas as atividades económicas, inclusive os serviços, quer estes sejam prestados por entidades privadas quer por entidades públicas. Dentro desta visão de “qualidade” de serviços, pretende este projeto de intervenção estudar a qualidade do serviço prestado, pelos serviços académicos de uma escola de ensino superior público e elaborar uma proposta de medidas de melhoria. Numa primeira fase estudámos a satisfação dos alunos face ao serviço prestado tendo sido recolhida a opinião de 120 alunos. De seguida analisámos os processos e procedimentos dos SAC e posteriormente elaborámos uma proposta de melhoria para obtermos um serviço que vá ao encontro das necessidades do seu público-alvo, correspondendo em tempo e formato às suas expectativas, por forma a obter qualidade contínua no serviço prestado. Durante a elaboração deste projeto conseguimos colocar em prática algumas das medidas propostas, sobre as quais obtivemos resultados e opiniões muito positivas.-Market globalization has transformed the reality in which our organizations live, thus creating an increasingly aggressive competition and the need to fight for a position in the market in which they are where "quality" has become an increasingly prominent factor. Quality goes far beyond a product. Nowadays, quality extends to education and even services. Quality concerns not only the industry and all economic activities including services, whether they are provided by private or by public entities. Within this vision of "quality" in services, this project aims to study the quality of the services provided by the academic department of a higher education school and develop a proposal for improvement measures. At the beginning we studied the extent to which students were satisfied with the service provided and we collected the opinions of 120 students. Then we examined the processes and procedures of SAC and subsequently drafted a proposal aiming to improve and to obtain a service which would meet the needs of our target audience, with time and format corresponding to their expectations, in order to achieve continuous quality in service. During the preparation of this project we were able to put some of the measures into practice, during obtained very positive results and opinions.

The History of Expansion of the Genus Bursaphelenchus (Nematoda: Aphelenchida: Parasitaphelenchidae)

Because of globalization and removal of geographical barriers, frequent biological invasions of introduced species become an urgent environmental problem. According to the Convention on Biological Diversity (CBD), precise identification of dangerous aggressive species at the early stages of their invasion to new regions is the most important component of the environmental control and monitoring. To resist the potential environmental hazard, the precise data are required on the current distribution and history of expansion of pests that are of global economic importance.