Comparative proteome analysis of triclabendazole response in the liver fluke, Fasciola hepatica.

Control of Fasciola hepatica infections of livestock in the absence of vaccines depends largely on the chemical triclabendazole (TCBZ) because it is effective against immature and adult parasites. Overdependence on a single drug and improper application is considered a significant factor in increasing global reports of fluke resistant to TCBZ. The mode(s) of action and biological target(s) of TCBZ are not confirmed, delaying detection and the monitoring of early TCBZ resistance. In this study, to further understand liver fluke response to TCBZ, the soluble proteomes of TCBZ-resistant and TCBZ-susceptible isolates of F. hepatica were compared with and without in vitro exposure to the metabolically active form of the parent drug triclabendazole sulphoxide (TCBZ-SO), via two-dimensional gel electrophoresis (2-DE). Gel image analysis revealed proteins displaying altered synthesis patterns and responses both between isolates and under TCBZ-SO exposure. These proteins were identified by mass spectrometry supported by a F. hepatica expressed sequence tag (EST) data set. The TCBZ responding proteins were grouped into three categories; structural proteins, energy metabolism proteins, and “stress” response proteins. This single proteomic investigation supported the reductionist experiments from many laboratories that collectively suggest TCBZ has a range of effects on liver fluke metabolism. Proteomics highlighted differences in the innate proteome profile of different fluke isolates that may influence future therapy and diagnostics design. Two of the TCBZ responding proteins, a glutathione transferase and a fatty acid binding protein, were cloned, produced as recombinants, and both found to bind TCBZ-SO at physiologically relevant concentrations, which may indicate a role in TCBZ metabolism and resistance.

Identifying additional patients with diabetic nephropathy using the UK primary care initiative

AIMS:
The aim of this study was to use general practice data to estimate the prevalence of diabetic nephropathy within the registered diabetes patients and examine variation in practice prevalence and management performance since introduction of this initiative.
METHODS:
Reported quality indicators from the Northern Ireland General Practice Quality and Outcomes Framework were analysed for diabetes and diabetic nephropathy prevalence and management in the period 2004-2008. Variation in prevalence at practice level was assessed using multiple linear regression adjusting for age, practice size, deprivation and glycaemic control.
RESULTS:
In 2006-2007, 57,454 (4.1%) adult diabetic patients were registered in the denominator population of 1.4 million compared with 51,923 (3.8%) in 2004-2005 (mean practice range 0.5-7.7%). Diabetic nephropathy prevalence was 15.1 and 11.5%, respectively (8688 and 5955 patients). Documented diabetic nephropathy prevalence showed marked variation across practices (range 0-100%) and was significantly negatively correlated with diabetes list size, albumin creatinine ratio testing rates and renin-angiotensin-aldosterone system blockade use and positively correlated with exception reporting rates. Specifically, for every increase in 100 diabetic patients to a register, documented diabetic nephropathy prevalence reduced by 40% (P=0.003). On the positive side, median albumin-creatinine ratio testing rates doubled to 82% compared with figures in the pre-Framework era.
CONCLUSIONS:
Implementation of the Northern Ireland General Practice Quality and Outcomes Framework has positively benefitted testing for diabetic nephropathy and increased numbers of detected patients in a short space of time. Large variation in diabetic nephropathy prevalence remains and is associated with diabetes registry size, screening and treatment practices, suggesting that understanding this variation may help detect and better manage diabetic nephropathy.

Human odontoblasts express functional thermo-sensitive TRP channels: implications for dentin sensitivity

Odontoblasts form the outermost cellular layer of the dental pulp where they have been proposed to act as sensory receptor cells. Despite this suggestion, evidence supporting their direct role in mediating thermo-sensation and nociception is lacking. Transient receptor potential (TRP) ion channels directly mediate nociceptive functions, but their functional expression in human odontoblasts has yet to be elucidated. In the present study, we have examined the molecular and functional expression of thermo-sensitive TRP channels in cultured odontoblast-like cells and in native human odontoblasts obtained from healthy wisdom teeth. PCR and western blotting confirmed gene and protein expression of TRPV1, TRPA1 and TRPM8 channels. Immunohistochemistry revealed that these channels were localised to odontoblast-like cells as determined by double staining with dentin sialoprotein (DSP) antibody. In functional assays, agonists of TRPV1, TRPA1 and TRPM8 channels elicited [Ca2+]i transients that could be blocked by relevant antagonists. Application of hot and cold stimuli to the cells also evoked rises in [Ca2+]i which could be blocked by TRP-channel antagonists. Using a gene silencing approached we further confirmed a role for TRPA1 in mediating noxious cold responses in odontoblasts. We conclude that human odontoblasts express functional TRP channels that may play a crucial role in mediating thermal sensation in teeth. Cultured and native human odontoblasts express functional TRP channels that may play a crucial role in mediating thermal sensation in teeth.

Human Dental Pulp Fibroblasts Express the “Cold-sensing” Transient Receptor Potential Channels TRPA1 and TRPM8

Introduction: Transient receptor potential (TRP) channels comprise a group of nonselective calcium-permeable cationic channels, which are polymodal sensors of environmental stimuli such as thermal changes and chemicals. TRPM8 and TRPA1 are cold-sensing TRP channels activated by moderate cooling and noxious cold temperatures, respectively. Both receptors have been identified in trigeminal ganglion neurones, and their expression in nonneuronal cells is now the focus of much interest. The aim of this study was to investigate the molecular and functional expression of TRPA1 and TRPM8 in dental pulp fibroblasts.
Methods: Human dental pulp fibroblasts were derived from healthy molar teeth. Gene and protein expression was determined by polymerase chain reaction and Western blotting. Cellular localization was investigated by immunohistochemistry, and TRP functionality was determined by Ca2+ microfluorimetry.
Results: Polymerase chain reaction and Western blotting showed gene and protein expression of both TRPA1 and TRPM8 in fibroblast cells in culture. Immunohistochemistry studies showed that TRPA1 and TRPM8 immunoreactivity co-localized with the human fibroblast surface protein. In Ca2+ microfluorimetry studies designed to determine the functionality of TRPA1 and TRPM8 in pulp fibroblasts, we showed increased intracellular calcium ([Ca2+]i) in response to the TRPM8 agonist menthol, the TRPA1 agonist cinnamaldehyde, and to cool and noxious cold stimuli, respectively. The responses to agonists and thermal stimuli were blocked in the presence of specific TRPA1 and TRPM8 antagonists.
Conclusions: Human dental pulp fibroblasts express TRPA1 and TRPM8 at the molecular, protein, and functional levels, indicating a possible role for fibroblasts in mediating cold responses in human teeth.

18F-fluorodeoxyglucose positron emission tomography/computed tomography-based radiotherapy target volume definition in non-small-cell lung cancer: delineation by radiation oncologists vs. joint outlining with a PET radiologist?

Purpose: F-18-Fluorodeoxyglucose positron emission tomography/computed tomography (PET/CT) has benefits in target volume (TV) definition in radiotherapy treatment planning (RTP) for non small-cell lung cancer (NSCLC); however, an optimal protocol for TV delineation has not been determined. We investigate volumetric and positional variation in gross tumor volume (GTV) delineation using a planning PET/CT among three radiation oncologists and a PET radiologist.