Development and validation of a gene expression test to identify hard-to-heal chronic venous leg ulcers.

BACKGROUND: Chronic venous leg ulcers pose a significant burden to healthcare systems, and predicting wound healing is challenging. The aim of this study was to develop a genetic test to evaluate the propensity of a chronic ulcer to heal. METHODS: Sequential refinement and testing of a gene expression signature was conducted using three distinct cohorts of human wound tissue. The expression of candidate genes was screened using a cohort of acute and chronic wound tissue and normal skin with quantitative transcript analysis. Genes showing significant expression differences were combined and examined, using receiver operating characteristic (ROC) curve analysis, in a controlled prospective study of patients with venous leg ulcers. A refined gene signature was evaluated using a prospective, blinded study of consecutive patients with venous ulcers. RESULTS: The initial gene signature, comprising 25 genes, could identify the outcome (healing versus non-healing) of chronic venous leg ulcers (area under the curve (AUC) 0·84, 95 per cent c.i. 0·73 to 0·94). Subsequent refinement resulted in a final 14-gene signature (WD14), which performed equally well (AUC 0·88, 0·80 to 0·97). When examined in a prospective blinded study, the WD14 signature could also identify wounds likely to demonstrate signs of healing (AUC 0·73, 0·62 to 0·84). CONCLUSION: A gene signature can identify people with chronic venous leg ulcers that are unlikely to heal.

The clinical significance and impact of interleukin 15 on keratinocyte cell growth and migration.

Chronic wounds represent a significant burden to health services and are associated with patient morbidity. Novel methods to diagnose and/or treat problematic wounds are needed. Interleukin (IL)-15 is a cytokine involved in a number of biological processes and disease states such as inflammation, healing and cancer progression. The current study explores the expression profile of IL-15 and IL-15 receptor α (IL-15Rα) in chronic wounds and its impact on keratinocytes. IL-15 and IL-15Rα expression were examined in healing and non-healing chronic wounds using qPCR and immunohistochemical analysis. The impact of recombinant IL-15 (rhIL-15) on human adult low calcium temperature (HaCaT) keratinocyte growth and migratory potential was further examined. IL-15 transcript expression was slightly, though non-significantly elevated in healing chronic wounds compared with non-healing chronic wounds. IL-15 protein staining was minimal in both subtypes of chronic wounds. By contrast, IL-15Rα transcript and protein expression were both observed to be enhanced in non-healing chronic wounds compared with healing chronic wounds. The treatment of HaCaT cells with rhIL-15 generally enhanced cell growth and promoted migration. Analysis with small molecule inhibitors suggested that the pro-migratory effect of rhIL-15 may be associated with ERK, AKT, PLCγ and FAK signalling. IL-15 may promote healing traits in keratinocytes and the differential expression of IL-15Rα is observed in chronic wounds. Together, this may imply a complex role for this interleukin in wound healing.

Tissue invasion and metastasis: Molecular, biological and clinical perspectives.

Cancer is a key health issue across the world, causing substantial patient morbidity and mortality. Patient prognosis is tightly linked with metastatic dissemination of the disease to distant sites, with metastatic diseases accounting for a vast percentage of cancer patient mortality. While advances in this area have been made, the process of cancer metastasis and the factors governing cancer spread and establishment at secondary locations is still poorly understood. The current article summarizes recent progress in this area of research, both in the understanding of the underlying biological processes and in the therapeutic strategies for the management of metastasis. This review lists the disruption of E-cadherin and tight junctions, key signaling pathways, including urokinase type plasminogen activator (uPA), phosphatidylinositol 3-kinase/v-akt murine thymoma viral oncogene (PI3K/AKT), focal adhesion kinase (FAK), ß-catenin/zinc finger E-box binding homeobox 1 (ZEB-1) and transforming growth factor beta (TGF-ß), together with inactivation of activator protein-1 (AP-1) and suppression of matrix metalloproteinase-9 (MMP-9) activity as key targets and the use of phytochemicals, or natural products, such as those from Agaricus blazei, Albatrellus confluens, Cordyceps militaris, Ganoderma lucidum, Poria cocos and Silybum marianum, together with diet derived fatty acids gamma linolenic acid (GLA) and eicosapentanoic acid (EPA) and inhibitory compounds as useful approaches to target tissue invasion and metastasis as well as other hallmark areas of cancer. Together, these strategies could represent new, inexpensive, low toxicity strategies to aid in the management of cancer metastasis as well as having holistic effects against other cancer hallmarks.

Differentiation of tumour-promoting stromal myofibroblasts by cancer exosomes.

Activation of myofibroblast rich stroma is a rate-limiting step essential for cancer progression. The responsible factors are not fully understood, but TGFß1 is probably critical. A proportion of TGFß1 is associated with extracellular nano-vesicles termed exosomes, secreted by carcinoma cells, and the relative importance of soluble and vesicular TGFß in stromal activation is presented. Prostate cancer exosomes triggered TGFß1-dependent fibroblast differentiation, to a distinctive myofibroblast phenotype resembling stromal cells isolated from cancerous prostate tissue; supporting angiogenesis in vitro and accelerating tumour growth in vivo. Myofibroblasts generated using soluble TGFß1 were not pro-angiogenic or tumour-promoting. Cleaving heparan sulphate side chains from the exosome surface had no impact on TGFß levels yet attenuated SMAD-dependent signalling and myofibroblastic differentiation. Eliminating exosomes from the cancer cell secretome, targeting Rab27a, abolished differentiation and lead to failure in stroma-assisted tumour growth in vivo. Exosomal TGFß1 is therefore required for the formation of tumour-promoting stroma.

CFTR suppresses tumor progression through miR-193b targeting urokinase plasminogen activator (uPA) in prostate cancer.

Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is expressed in the epithelial cells of a wide range of organs/tissues from which most cancers are derived. Although accumulating reports have indicated the association of cancer incidence with genetic variations in CFTR gene, the exact role of CFTR in cancer development and the possible underlying mechanism have not been elucidated. Here, we report that CFTR expression is significantly decreased in both prostate cancer cell lines and human prostate cancer tissue samples. Overexpression of CFTR in prostate cancer cell lines suppresses tumor progression (cell growth, adhesion and migration), whereas knockdown of CFTR leads to enhanced malignancies both in vitro and in vivo. In addition, we demonstrate that CFTR knockdown-enhanced cell proliferation, cell invasion and migration are significantly reversed by antibodies against either urokinase plasminogen activator (uPA) or uPA receptor (uPAR), which are known to be involved in various malignant traits of cancer development. More interestingly, overexpression of CFTR suppresses uPA by upregulating the recently described tumor suppressor microRNA-193b (miR-193b), and overexpression of pre-miR-193b significantly reverses CFTR knockdown-enhanced malignant phenotype and abrogates elevated uPA activity in prostate cancer cell line. Finally, we show that CFTR gene transfer results in significant tumor repression in prostate cancer xenografts in vivo. Taken together, the present study has demonstrated a previously undefined tumor-suppressing role of CFTR and its involvement in regulation of miR-193b in prostate cancer development.

Colostral antibody protection and interference with immunity in lambs born from sheep vaccinated with an inactivated Bluetongue serotype 8 vaccine.

Widespread vaccination programmes against Bluetongue virus serotype 8 (BTV-8), using inactivated vaccines, are being carried out across many countries in northern, western and southern Europe. This study investigates the extent and length of colostral antibody protection, as well as the degree of colostral antibody induced interference of the immune response to BTV-8, in sheep. Significantly lower titres of neutralising antibodies were transferred in colostrum to lambs born from sheep vaccinated once as opposed those vaccinated twice (single vaccine in the first year and a booster vaccine in the second year). On BTV-8 challenge, lambs born from sheep vaccinated on two occasions, with the second booster vaccine given approximately 1 month prior to lambing, were protected from clinical disease for up to 14 weeks. BTV-8 was isolated from 5 of the 22 challenged lambs, although only one of these lambs showed a transient rise in body temperature with no other clinical signs. Lambs born from ewes given a second booster vaccine 1 month prior to lambing, are likely to be protected from clinical disease for at least 14 weeks, whereas lambs born from ewes vaccinated once are likely to be protected for a shorter time. Colostral antibodies present in the 13-14-week-old lambs appeared to interfere with the humoral response to challenge virus. These results suggest that colostral antibodies may interfere with vaccination in lambs up to at least 14 weeks of age.

Seroconversion, neutralising antibodies and protection in bluetongue serotype 8 vaccinated sheep.

Bluetongue virus serotype 8 (BTV-8) has caused a major outbreak of disease in cattle and sheep in several countries across northern and western Europe from 2006 to 2008. In 2008 the European Union instigated a mass-vaccination programme in affected countries using whole virus inactivated vaccines. We evaluated vaccinal responses in sheep and the ability of the vaccine to protect against experimental challenge. Sheep vaccinated 10 months previously under field conditions were challenged with BTV-8. One of 7 vaccinated sheep became infected, as evidenced by detection of viral RNA by real-time RT-PCR and by virus isolation. The remaining 6 sheep appeared fully protected from virus replication. None of the vaccinated sheep showed clinical signs of BTV and there was a good correlation between the presence of neutralising antibodies on challenge and protection. Commercially available ELISAs were evaluated for their ability to detect antibodies in sheep vaccinated on a single occasion. The sandwich (double antigen) ELISA assays were found to be more sensitive at detecting antibodies in vaccinated sheep than the competitive ELISAs.

Bluetongue virus: European Community proficiency test (2007) to evaluate ELISA and RT-PCR detection methods with special reference to pooling of samples.

Bluetongue virus European Community national reference laboratories (BTV-EC-NRLs) participated in an inter-laboratory proficiency test in 2007. The aim of the inter-laboratory proficiency test was to determine the ability of laboratories to detect antibodies to a series of BTV serotypes by cELISA and to detect viral RNA in animals infected with the European strain of BTV-8 by RT-PCR. Both serum and EDTA blood sample were diluted in order to determine the sensitivity of the assays. All the cELISAs were 'fit-for purpose' to detect antibodies to the common BTV serotypes circulating in Europe and the real time RT-PCR assays were all capable of detecting BTV-8 RNA albeit with varying sensitivities. There were however inconsistencies in the ability of the gel-based PCR assays to detect BTV RNA. In addition, samples taken on the first day of viraemia and at the peak of viraemia from animals experimentally infected with BTV-8, were diluted to determine if the diluting of samples affected the ability of the Shaw et al. (Shaw, A.E., M., P., Alpar, H.O., Anthony, S., Darpel, K.E., Batten, C.A., Carpenter, S., Jones, H., Oura, C.A.L., King, D.P., Elliott, H., Mellor, P.S., Mertens, P.P.C., 2007. Development and validation of a real-time RT-PCR assay to detect genome bluetongue virus segment 1. Journal of Virological Methods) RT-PCR assay to detect BTV-RNA at these time-points. Results indicated that, if samples were taken at the onset of viraemia, diluting at 1/5 resulted in a reduced ability of the assay to detect BTV RNA in the diluted compared to the neat samples. Diluting samples taken at the peak of viraemia at 1/10 however resulted in no loss in sensitivity.

Validation of open-surgery VR trainer.

VREST (Virtual Reality Educational Surgical Tools) is developing a universal and autonomous simulation platform which can be used for training and assessment of medical students and for continuing education of physicians. With the VREST - Virtual Lichtenstein Trainer, simulating the open surgery procedure of the inguinal hernia repair according to Lichtenstein, the validation of the simulator is ongoing. Part of this trajectory is the evaluation of the transfer of training of the virtual incision making. One group of students trained incision making on the VREST platform where the control group did not. In an experiment both groups has to perform several incision tasks on a manikin. The results are not available yet but will be presented at the MMVR14 conference.

Open surgery in VR: inguinal hernia repair according to Lichtenstein.

VREST (Virtual Reality Educational Surgical Tools) is developing a universal and autonomous simulation platform which can be used for training and assessment of medical students and for continuing education of physicians. A workstation consisting of two haptic devices and a 3D vision system is part of the VREST platform. Another part of the platform is a generic software environment in which lessons can be built by the teacher and performed by their students. Using the platform one can see, feel and decide as in reality. With the assessment tool the progress and skills of the students can be supervised. The first lesson build on the VREST platform is an inguinal hernia repair according to Lichtenstein. This is an open surgery procedure. The VREST platform is used prior to the first operating room surgery of the resident. Interactive models and case dependent feedback is used to enlarge the residents' cognition. This should reduce the training time in the operating room.

The VREST learning environment.

The VREST learning environment is an integrated architecture to improve the education of health care professionals. It is a combination of a learning, content and assessment management system based on virtual reality. The generic architecture is now being build and tested around the Lichtenstein protocol for hernia inguinalis repair.

Applicability of an image analysis system in alveolar bone loss measurement.

Clinical and epidemiological studies in the field of periodontics and endodontics often utilize radiographs to monitor or measure the changes in bone structure and density. Periodontal bone loss or gain can be quantified on a radiograph by measurement of the distance between the bottom of the bony pocket and the apical contour of the involved tooth. The objective of this investigation was to study the accuracy of an image analysis system (IAS) to measure changes in height of the interproximal crest on radiographs. Artificial bone lesions were introduced in a dissectioned part of a human mandible. The distances between crest and apices were measured with a micrometer (MM). Radiographs were produced with horizontal and vertical deviations of 10 degrees. The radiographs were digitized and processed by computer. The landmarks in the digital image were enhanced mathematically and by histogram-based thresholding. The depth of the introduced defect was increased 6 times, followed by the measurement procedure. The IAS produced measurements of crown-apex distances with an accuracy of 0.066 to 0.358 mm. Repeated crest height measurements were recorded with an accuracy of 0.112 to 0.184 mm. Both the histogram-based binarization and the ellipse-fitting type of contour detection could be applied precisely. Misangulation errors during radiographic exposure of 10 horizontal or vertical did not statistically significant influence the IAS-measurements. The IAS can be applied in clinical trials and follow-up studies.

A computer-aided image analysis system for area measurement of tooth root surfaces.

The characteristics of a computer-aided Image Analysis System (IAS) to conduct linear and area measurements on tooth root surfaces were investigated. The borders of the features to be measured were extracted from a digitized video image by histogram-based thresholding and binarization. The lengths and square areas were expressed in the number of pixels. The reference-length and area of one pixel were obtained through measurement of the diameter of a metal ball of known diameter in the same digital image. The validity of the IAS was tested using radiographs of teeth containing round metal restorations of known diameter and area. The accuracy of the length measurements ranged from -2.0 to 2.1%, whereas the accuracy of area measurement was between -5.3 and 0.8%. The precision of the IAS to identify the borders of a tooth root surface and to reproducibly calculate its area, expressed in a coefficient of variation, ranges from 0.49 to 4.11%. The results indicate that imaging techniques can be applied to obtain very accurate measurements of tooth root surfaces.

Towards the legalization of African folk therapy.

'Folk therapy' is distinguished from western scientific medical practice, and the role of the former is defined in its context. 'Diviners', 'medicinemen', 'witches' and 'sorcerers' are defined and distinguished. The colonial influence and its legacies in Africa are examined, as are the post-colonial adjustments. The crucial question is whether folk therapy should be legally recognized and controlled. The conclusion reached is that this might be a premature, and therefore detrimental, step at present, as it might endanger the usefulness and survival of a valuable cultural heritage.