Heterogeneity and clinical significance of ETV1 translocations in human prostate cancer

A fluorescence in situ hybridisation (FISH) assay has been used to screen for ETV1 gene rearrangements in a cohort of 429 prostate cancers from patients who had been diagnosed by trans-urethral resection of the prostate. The presence of ETV1 gene alterations (found in 23 cases, 5.4%) was correlated with higher Gleason Score (P=0.001), PSA level at diagnosis (P=<0.0001) and clinical stage (P=0.017) but was not linked to poorer survival. We found that the six previously characterised translocation partners of ETV1 only accounted for 34% of ETV1 re-arrangements (eight out of 23) in this series, with fusion to the androgen-repressed gene C15orf21 representing the commonest event (four out of 23). In 5'-RACE experiments on RNA extracted from formalin-fixed tissue we identified the androgen-upregulated gene ACSL3 as a new 5'-translocation partner of ETV1. These studies report a novel fusion partner for ETV1 and highlight the considerable heterogeneity of ETV1 gene rearrangements in human prostate cancer.

Function of host defence peptide LL-37 in human periodontal disease.

Objectives: Fibroblasts play a significant role as regulators of the host response in periodontal disease, responding to bacterial stimulation by producing an array of inflammatory cytokines and chemokines. LL-37, a host defence peptide, inhibits LPS-induced cytokine signalling in macrophages, suggesting an immunomodulatory role. The objective was to investigate the interaction between LL-37 and gingival fibroblasts – both its direct regulation of fibroblast activity and its effect on fibroblast response to LPS activation. Methods: Human gingival fibroblasts (HGFs) were incubated for 24 hours in the presence of either P. gingivalis LPS (10µg/ml) or E. coli LPS (10ng/ml) along with LL-37 (0-50 µg/ml). IL-6 and IL-8 production by HGFs in the conditioned medium was determined by ELISA. Western blot was performed to determine the effect of LL-37 on LPS -induced IκBα degradation in HGFs following LPS stimulation over 2 hours. DNA microarray analysis was performed on cell populations incubated for 6 hr in the presence or absence of the peptide. Confirmation of LL-37 effects on specific gene expression was obtained by QPCR. Results: At low concentrations (≤ 5 µg/ml) LL-37 significantly inhibited LPS-induced cytokine production by HGFs. At higher concentrations LL-37 induced IL-8 production independent of LPS. Addition of LL-37 blocked LPS-induced IκBα degradation in HGFs. Microarray analysis revealed that LL-37 (50µg/ml) upregulated a significant number of cytokines and chemokines by > 5 fold. Upregulation of five of these, CXCL1, CXCL2, CXCL3, IL-24 and IL-8 was confirmed by Q-PCR. Conclusion: The host defence peptide LL-37, the only known human cathelicidin, appears to have pleiotrophic effects in innate immunity. At least some of these are mediated through cytokine and chemokine signalling networks. The ability of LL-37 to reduce bacterial LPS-induced cytokine production in gingival fibroblasts, at low concentrations, suggests a potential therapeutic role in the management of periodontal disease.

Data Cleaning and Outlier Removal: Application in Human Skin Detection

An outlier removal based data cleaning technique is proposed to
clean manually pre-segmented human skin data in colour images.
The 3-dimensional colour data is projected onto three 2-dimensional
planes, from which outliers are removed. The cleaned 2 dimensional
data projections are merged to yield a 3D clean RGB data. This data
is finally used to build a look up table and a single Gaussian classifier
for the purpose of human skin detection in colour images.

TRP channel expression in human gingival fibroblasts

Background: The transient receptor potential (TRP) super family of ion channels is believed to play a critical role in sensory physiology, acting as transducers for thermal, mechanical and chemical stimuli. Our understanding of the role of TRP channel expression in gingival fibroblasts is currently limited. The role of non-neuronal TRP channel expression is an area of much research interest particularly since TRP channel activation has recently been hypothesised to be associated with inflammation. Objectives: The present study was designed to determine the expression of TRPV1, TRPV2, TRPV3 and TRPV4 on human gingival fibroblasts. Methods: Human gingival fibroblasts were derived by explant culture from surgical tissue following ethical approval. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM), containing 10% fetal calf serum (FCS) in 5% CO2. Cell lysates of gingival fibroblasts were electrophoresed and blotted on to nitrocellulose before probing with specific anti-TRP antibodies. Immunoreactive bands were detected using anti-species antibodies and chemiluminescent detection. Results: Gingival fibroblasts were shown to express proteins corresponding to the TRPV1, TRPV2, TRPV3 and TRPV4 channels as determined by western blotting. Conclusion: This study reports for the first time the expression of TRPV1, TRPV2, TRPV3 and TRPV4 by gingival fibroblasts. Knowledge of the expression of TRP channels by human gingival fibroblasts will guide future research on the roles of TRP channels in sensing the external environment in the oral cavity.

Reference gene selection in human periodontal ligament cells

Background: Real-time quantitative PCR (qPCR) is a highly sensitive and specific method which is used extensively for determining gene expression profiles in a variety of cell and tissue types. In order to obtain accurate and reliable gene expression quantification, qPCR data are generally normalised against so-called reference or housekeeping genes. Ideally, reference genes should have abundant and stable RNA transcriptomes under the experimental conditions employed. However, reference genes are often selected rather arbitrarily and indeed some have been shown to have variable expression in a variety of in vitro experimental conditions.
Objective: The objective of the current study was to investigate reference gene expression in human periodontal ligament (PDL) cells in response to treatment with lipopolysaccharide (LPS).
Method: Primary human PDL cells were grown in Dulbecco’s Modified Eagle Medium with L-glutamine supplemented with 10% fetal bovine serum, 100UI/ml penicillin and 100µg/ml streptomycin. RNA was isolated using the RNeasy Mini Kit (Qiagen) and reverse transcribed using the QuantiTect Reverse Transcription Kit (Qiagen). The expression of a total of 19 reference genes was studied in the presence and absence of LPS treatment using the Roche Reference Gene Panel. Data were analysed using NormFinder and Bestkeeper validation programs.
Results: Treatment of human PDL cells with LPS resulted in changes in expression of several commonly used reference genes, including GAPDH. On the other hand the reference genes β-actin, G6PDH and 18S were identified as stable genes following LPS treatment.
Conclusion: Many of the reference genes studied were robust to LPS treatment (up to 100 ng/ml). However several commonly employed reference genes, including GAPDH varied with LPS treatment, suggesting they would not be ideal candidates for normalisation in qPCR gene expression studies.

TNF-α Sensitizes Mechanoreceptors in Human Odontoblasts

Background: Mechanotransduction in the dental pulp is mediated by mechano-sensitive trigeminal afferents but accumulating evidence suggests odontoblasts also contribute to mechano-sensory functions of the pulp as evidenced by expression of TRP channels, calcium-activated potassium channels and TREK-1 potassium channels. Activation of these mechano-sensitive channels is considered critical for the mechanotransduction of fluid movement within dentinal tubules into electrical signals transmitted by the pulpal afferents to elicit tooth sensitivity and pain. Since tooth pain and sensitivity are potentiated by inflammation we hypothesise that the inflammatory cytokine TNF-α sensitizes odontoblast responses to mechanical stimuli. Objective: To investigate the effect of TNF-α on the response of odontblast-like cells to mechanical stimuli. Method: Odontoblast-like cells were derived from dental pulp cells of immature third molars as previously described (El-karim et al 20112011 Pain, 152, 2211-2223). Odontoblast response to mechanical stimuli (application of hypotonic solution) was determined using ratiometric calcium imaging. Cells were treated with TNF-α for either 24hrs or short application for 10 mins prior to calcium imaging. Result: Odontoblast-like cells responded to hypotonic solution (230 mOSM) by increase in cytoplasmic Ca2+ concentration [Ca+2]i that was reduced to near base line in the presence of the TRPV4 antagonist RN-1734. Incubation of odontoblast -like cells with TNFα for 24 hrs resulted in a significant increase in cytoplasmic Ca2+ concentration in response to hypotonic stimuli compared to untreated cells. Similar results were obtained when cells were treated with TNF-α for 10 mins prior to imaging. Conclusion: Both short and long term treatment of odontoblasts-like cells with TNF-α resulted in enhanced responses to mechanical stimuli mediated via TRPV4 channel suggesting a role for this channel in inflammatory dental pain.

NPY Y1 and VPAC1 receptor expression in human dental pulps

Neuropeptides such as neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) have been shown by our research group to be present in human dental pulp tissue. Neuropeptides cannot cross cell membranes and therefore to exert their biological effects they must bind to selected receptors on the surface of target cell membranes. However, the expression of receptor proteins for NPY and/or VIP have yet to be reported in human pulp tissue. The presence of neuropeptide receptors can be conveniently determined by Western blotting using specific anti-receptor antibodies. Objectives: The aim of this work was to identify the presence of the NPY Y1 receptor and the VIP receptor VPAC1 in human dental pulp tissue from both intact and carious teeth using Western blotting. Methods: Pulp tissue was collected from both intact and carious teeth and membrane preparations from these tissues were then subject to sodium dodecyl sulphate gel electrophoresis (SDS-PAGE), transferred to nitrocellulose and probed with specific antibodies to either the NPY Y1 receptor or the VPAC1 receptor. Results: Individual Western blotting experiments revealed the presence of immunoreactive bands corresponding to the known molecular weights of the NPY Y1 and VPAC1 receptor proteins in both intact and carious pulp samples. Conclusions: Demonstration of the presence of NPY Y1 and VPAC1 receptor protein expression in pulpal tissue from intact and carious teeth provides further support for the roles of these neuropeptides in pulpal health and disease.

Timing of response differentiation in human motor cortex during a speeded Go/No-go task

We explored the brain's ability to quickly prevent a pre-potent but unwanted motor response. To address this, transcranial magnetic stimulation was delivered over the motor cortex (hand representation) to probe excitability changes immediately after somatosensory cues prompted subjects to either move as fast as possible or withhold movement. Our results showed a difference in motor cortical excitability 90 ms post-stimulus contingent on cues to either promote or prevent movement. We suggest that our study design emphasizing response speed coupled with well-defined early probes allowed us to extend upon similar past investigations into the timing of response inhibition.

'Heat-sensing' TRP channel expression in human odontoblasts

Background: Thermal changes in the oral cavity are a common trigger of dental pain. Several members of the transient receptor potential (TRP) super family of ion channels are believed to play a critical role in sensory physiology, where they act as transducers for thermal, mechanical and chemical stimuli. Objectives: The present study was designed to determine the expression and functionality of the TRPV1 channel in human odontoblasts. Methods: Cultured human odontoblasts were derived from dental pulp cells induced with 2 mM beta-glycerophosphate. Molecular and protein expression of TRPV1 was confirmed by PCR, western blotting and immunohistochemistry. Functional expression of the ‘heat-sensing' TRPV1 channel was investigated using a Ca2+ microfluorimetry assay in the presence of agonists/antagonists or with appropriate adjustment of the recording chamber temperature. Results: The odontoblastic phenotype of the cells was confirmed by the expression of the odontoblast markers dentin sialophosphoprotein (DSPP) and nestin. Expression of TRPV1 in human odontoblastic cells was confirmed by PCR, western blotting and immunohistochemistry. Odontoblasts were shown to respond to pharmacological agonists and to increasing temperature by an increase in intracellular Ca2+. Both the pharmacological and temperature responses could be blocked by specific antagonists. These results indicate that odontoblasts may sense heat via TRPV1. Conclusion: This study reports that TRPV1 is expressed by human odontoblasts and is activated by specific pharmacological agonists and by heat.
This work was supported by Research Grants from the Royal College of Surgeons of Edinburgh and the British Endodontic Society

An in vitro model of nociceptors in human trigeminal nerves

Background: The oro-facial region is densely innervated by the trigeminal nerve, which when stimulated can induce noxious pain sensation and contribute to neurogenic inflammation in local tissues. Recent research on the expression of specialised ion channels on the trigeminal nerve has highlighted the need to undertake more extensive studies on ion channel expression/functionality with the aim of elucidating their role in pain sensations. A major family of such ion channels is the transient receptor potential (TRP) channels which are activated by a wide variety of thermal, mechanical or chemical stimuli and merit investigation as possible druggable targets for future analgesics.
Objective: Study of TRP channel expression and regulation in oro-facial tissues is hindered by the fact that the cell bodies of neurons innervating these tissues are located in the trigeminal ganglion. Using dental pulp stem cells differentiated towards peripheral neuronal equivalents (PNEs), we sought to determine TRP channel expression, functionality and potential modulation by cytokines in this novel model.
Method: Dental pulp stem cells (DPSCs) were grown on substrate-coated tissue culture plates and differentiated towards a neuronal phenotype using neuronal induction media. Quantitative polymerase chain reaction (qPCR) was performed on PNEs +/-cytokine treatment. Ion channel functionality was investigated using whole cell patch clamping.
Result: qPCR analysis showed that PNEs expressed the TRP channels TRPA1, TRPV1, TRPV4 and TRPM8. TRPA1 was the most abundantly expressed TRP channel studied whereas TRPM8 was lowly expressed. TRP channel expression was shown to be regulated by treatment with inflammatory cytokines. Patch clamp studies using specific agonists and antagonists for TRPA1 and TRPV1 showed these channels were functional.
Conclusion: PNEs differentiated from DPSCs provide a suitable model for TRP channel expression, regulation, and sensistisation in oro-facial tissues. This human neuronal model has potential for use in pre-clinical studies of novel analgesics.

Functional expression of thermo-sensitive TRP channels in human odontoblasts

Introduction: Accumulating evidence supports a role for odontoblasts in initiating tooth pain, however direct ionic mechanisms underlying dentine nociceptive function remain unclear. The transient receptor potential (TRP) ion channels are directly related to cellular mechanisms of nociception and thermo-sensitive function but their expression by human odontoblasts remains to be determined. Objectives: To investigate the expression and functionality of the thermo-sensitive TRP channels TRPV1, TRPV4, TRPM8 and TRPA1 in human odontoblasts. Methods: Human odontoblasts were derived from dental pulp of immature permanent third molars by explant method. Cell lysates of odontoblasts were subject to SDS- polyacrylamide gel electrophoresis and proteins were blotted onto nitrocellulose membranes. Blots were probed with primary antibodies to TRPA1, TRPM8, TRPV4 and TRPV1. Detection of bound primary antibodies was achieved using appropriate anti-species antibody conjugates and chemiluminescent substrates. Functionality of the channels was determined with Ca2+ microfluorimetry, where cells grown in cover slips and incubated with Fura 2AM prior to stimulation with capsaicin (TRPV1 agonist), 4 alpha-phorbol 12,13-didecanoate (4áPDD) (TRPV4 agonist), icilin (TRPA1 agonist) and menthol (TRPM8 agonist). Emitted fluorescence was measured and the fluorescence ratio (R) was calculated as F340/F380 to determine the level of [Ca2+]i. Results: Western blotting confirmed the molecular localisation of thermo-sensitive TRP channels in human odontoblasts. Functionality assays revealed increase in [Ca2+]i in response to capsacin, icillin, methanol and 4áPDD indicating functional expression of TRPV1, TRPA1, TRPM8 and TRPV4 respectively. Conclusions: Functional expression of thermo-sensitive TRP channels in human odontoblasts may indicate a crucial role for odontoblasts in thermally induced dental pain. (Supported by a Research Grant from the Royal College of Surgeons of Edinburgh)

Co-expression of NPY and VIP in human dental pulp

Introduction: The regulation of pulpal haemodynamics in health and disease involves sympathetic and parasympathetic mechanisms in which both neuropeptide Y (NPY; a sympathetic vasoconstrictor) and vasoactive intestinal polypeptide (VIP; a parasympathetic vasodilator) may play potential pathophysiological roles. We have previously investigated the levels of NPY or VIP present in human dental pulp tissue and shown that their expression is up-regulated in caries induced pulpal inflammation. Objectives: The aim of this study was to investigate the potential correlation between NPY and VIP levels measured in the same dental pulp samples using radioimmunoassay (RIA). Methods: Pulp tissue was obtained from extracted teeth, classified as follows; healthy (n=22), moderately carious (n=20) and grossly carious (n=26). Samples were processed for RIA by boiling in acetic acid as previously described. The levels of NPY and VIP, measured by RIA, were expressed as ng/gram of pulp tissue. The nature of the relationship between NPY and VIP levels in human pulp tissue was tested by calculating Pearson's product moment correlation coefficient using the linear regression test. Results: Calculation of Pearson product moment correlation coefficient showed a significant negative correlation between NPY and VIP levels in pulp tissue samples from non-carious teeth (p = 0.02, r = -.48). This negative correlation in non-carious teeth changed to a significant positive correlation in carious teeth when the levels of NPY and VIP were compared (p = 0.03, r= 0.311). Conclusions: In non-carious teeth, the negative correlation between NPY and VIP levels is in keeping with the previously described modulatory influence of cholinergic nerves on sympathetic function which may be perturbed as caries develops.

Functional expression of TRPV4 in human periodontal ligament cells

Background: Periodontal ligament (PDL) cells are exposed to physical forces in vivo in response to mastication, parafunction, speech and orthodontic tooth movement. Although it has been shown that PDL cells perceive and respond directly to mechanical stimulation, the nature of the ion channels that mediate this mechanotransduction remain to be fully elucidated. The transient receptor potential (TRP) superfamily of ion channels is believed to play a critical role in sensory physiology, where they act as transducers for thermal, chemical and mechanical stimuli. Recent studies have shown that members of the vanilloid (TRPV) and ankyrin (TRPA) subfamilies encode mechanosensitive TRPs. The vanilloid family member TRPV4 is one such non selective calcium permeable cationic channel which has been shown to be activated by chemical ligands, hypotonicity, and mechanical stimuli. Objectives: The objective of the current study was to investigate functional expression of TRPV4 in cultured human PDL cells. Methods: Human PDL cells were grown in Dulbecco's Modified Eagle Medium with L-glutamine supplemented with 10% fetal bovine serum (FBS), 100UI/ml penicillin and 100μg/ml streptomycin. Cells in passage 4-6 were used in all experiments. TRPV4 functional expression was determined using ratiometric calcium imaging. Cultured cells were loaded with intracellular Ca2+ probe fura-2 and cells were then stimulated with the TRPV4 agonists, 4alpha-phorbol 12,13-didecanoate (4alpha-PDD), GSK1016790A or hypotonic solution. The TRPV4 antagonist RN 1734 was used to block the corresponding agonist responses. Results: PDL fibroblasts responded to application of TRPV4 agonists and hypotonic stimuli by an increase in intracellular calcium which was attenuated in the presence of the TRPV4 antagonist. Conclusions: We have shown for the first time the functional expression of the mechanosensitive TRPV4 channel in human PDL cells. The molecular identity and mechanisms of activation of mechanosensitive TRP channels in PDL cells merit further investigation.

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.

Vasoactive Intestinal Polypeptide (VIP) in Human Dental Pulps

Pulpal innervation is not exclusively sensory and there are potential roles for other neuropeptides such as vasoactive intestinal polypeptide (VIP) in pulpal health and disease. In the systemic circulation VIP relaxes vascular smooth muscles leading to vasodilatation. It has been shown that VIP fibres are associated with pulpal blood vessels and therefore VIP may mediate vasoactivity in the dental pulp. A growing body of evidence has now demonstrated that an additional major physiological role of VIP is to act as a survival factor. In order to gain a better understanding of the role of neuropeptides in the caries process it is of interest to specifically examine a role for VIP. Objectives: The aim of the present study was to determine the levels of VIP in carious (moderately carious and grossly carious) compared with non-carious teeth. Methods: A total of 68 teeth were included in the study (22 non-carious, 20 moderately carious and 26 grossly carious). VIP was measured in all samples using a sensitive and specific radioimmunoassay. Results: The mean concentration of VIP in the pulps of non-carious teeth was 7.69 ng/g (9.41 SD) compared to 14.93 ng/g (15.58 SD) in carious teeth. Pair-wise comparisons of VIP levels using Tukey’s test showed statistically significant differences in VIP expression between non-carious and moderately carious teeth (p=0.002) and between moderately and grossly carious teeth, (p=0.002). Conclusion: The significantly increased levels of VIP in moderately carious pulps compared with either non-carious or grossly carious pulps may suggest a role for VIP as a protective or survival factor.