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Proof-of-principle Study (proof-of-principle + study)
Selected AbstractsImage-processing chain for a three-dimensional reconstruction of basal cell carcinomas,EXPERIMENTAL DERMATOLOGY, Issue 7 2010Patrick Scheibe Please cite this paper as: Image-processing chain for a three-dimensional reconstruction of basal cell carcinomas. Experimental Dermatology 2010; 19: 689,691. Abstract:, Basal cell carcinoma (BCC) is the most common malignant skin cancer. For a deeper insight into the specific growth patterns of the tumorous tissue in BCC, we have focused on the development of a novel automated image-processing chain for 3D reconstruction of BCC using histopathological serial sections. For fully automatic delineation of the tumor within the tissue, we apply a fuzzy c-means segmentation method. We used a novel multi-grid form of the non-linear registration introduced by Braumann and Kuska in 2005 effectively suppressing registration runs into local minima (possibly caused by diffuse nature of the tumor). Our method was successfully applied in a proof-of-principle study for automated reconstruction. [source] Periodontal repair in dogs: space-providing ePTFE devices increase rhBMP-2/ACS-induced bone formationJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 8 2003Ulf M.E. Wikesjö Abstract Background: Recombinant human bone morphogenetic protein-2 (rhBMP-2) technologies have been shown to enhance alveolar bone formation significantly. Biomaterial (carrier) limitations, however, have restricted their biologic potential for indications where compressive forces may limit the volume of bone formed. The objective of this proof-of-principle study was to evaluate the potential of a space-providing, macroporous ePTFE device to define rhBMP-2-induced alveolar bone formation using a discriminating onlay defect model. Methods: Routine, critical size, 5,6 mm, supra-alveolar, periodontal defects were created around the third and fourth mandibular premolar teeth in four young adult Hound Labrador mongrel dogs. All jaw quadrants received rhBMP-2 (0.4 mg) in an absorbable collagen sponge (ACS) carrier. Contralateral jaw quadrants in subsequent animals were randomly assigned to receive additionally the dome-shaped, macroporous ePTFE device over the rhBMP-2/ACS implant or no additional treatment. The gingival flaps were advanced to cover the ePTFE device and teeth, and sutured. Animals were scheduled for euthanasia to provide for histologic observations of healing at 8 weeks postsurgery. Results: Healing was uneventful without device exposures. New bone formation averaged (±SD) 4.7±0.2 mm (98%) and 4.5±0.4 mm (94%) of the defect height, respectively, for jaw quadrants receiving rhBMP-2/ACS with the ePTFE device or rhBMP-2/ACS alone (p>0.05). In contrast, the regenerated bone area was significantly enhanced in jaw quadrants receiving rhBMP-2/ACS with the ePTFE device compared to rhBMP-2/ACS alone (9.3±2.7 versus 5.1±1.1 mm2; p<0.05). Cementum formation was similar for both treatment groups. Ankylosis compromised periodontal regeneration in all sites. Conclusions: The results suggest that the novel space-providing, macroporous ePTFE device appears suitable as a template to define rhBMP-2/ACS-induced alveolar bone formation. Zusammenfassung Hintergrund: Es wurde gezeigt, dass das rekombinante menschliche knochenmorphogenetische Protein 2 (rhBMP-2) die alveoläre Knochenbildung signifikant erhöht. Limitationen des Biomaterials (Träger) haben jedoch die biologischen Potenzen des Materials für die Indikationen, wo komprimierende Kräfte das Volumen des zu bildenden Knochen limitierten, eingeengt. Das Ziel dieser prinzipiellen geprüften Studie war die Evaluation der Platzhalterfunktion einer makroporösen e-PTFE Membran, um die von rhBMP-2 induzierten Knochenbildung unter Nutzung eines differenzierenden Onlaydefektmodells zu definieren. Methoden: Routinemäßig wurden supraalveoläre parodontale Defekte mit der kritischen Größe von 5,6 mm um die dritten und vierten Prämolaren bei 4 jungen adulten Labrodormischhunden geschaffen. Alle Quadranten erhielten rhBMP-2 (0.4 mg) in einem resorbierbaren Kollagenschwamm (ACS). Kontralaterale Quadranten bei den aufeinander folgenden Tieren wurden zufällig ausgewählt, um zusätzlich eine domförmige makroporöse e-PTFE Membran über das rhBMP-2/ACS Implantat oder keine zusätzliche Therapie zu erhalten. Die gingivalen Lappen wurden so präpariert, dass sie die e-PTFE Membran und Zähne bedeckten und vernäht. Die Tiere wurden 8 Wochen nach der Operation getötet und für histologische Untersuchungen vorbereitet. Ergebnisse: Die Heilung war komplikationslos ohne Exposition der Membran. Die neue Knochenbildung betrug durchschnittlich (±SD) 4.7±0.2 mm (98%) und 4.5±0.4 mm (94%) der Defekthöhe für die Quadranten, die rhBMP-2/ACS mit der e-PTFE Membran erhielten oder rhBMP-2/ACS allein (p>0,05). Im Kontrast dazu war das regenerierte Knochenfeld signifikant erweitert bei den Kieferquadranten, die rhBMP-2/ACS mit e-PTFE Membran erhielten im Vergleich zu denjenigen mit rhBMP-2/ACS allein (9.3±2.7 vs. 5.1±1.1 mm2; p<0.05). Die Zementbildung war in beiden Behandlungsgruppen ähnlich. Ankylosen gefährdeten die parodontalen Regeneration in allen Flächen. Schlussfolgerungen: Die Ergebnisse zeigen, dass die neue makroporöse Platzhalter e-PTFE Membran als Schablone nützlich ist, um die rhBMP-2/ACS induzierte alveoläre Knochenbildung zu betonen. Résumé Contexte: Des technologies utilisant la protéine-2 osseuse morphogénétique humaine recombinée (rhBMP-2) ont montré qu'elle permettait d'augmenter significativement la formation d'os alvéolaire. Les limites du biomatériel (vecteur), cependant, ont restreint leur potentiel biologique aux indications pour lesquels des forces compressives pourraient limiter le volume d'os en formation. L'objectif de cette étude fut d'évaluer le potentiel d'un dispositif en ePTFE macro-poreux permettant de créer un espace pour définir la formation d'os alvéolaire induit par la rhBMP-2 en utilisant un modèle discriminatoire de lésion. Méthodes: Des lésions parodontales supra-alvéolaires de taille critique, 5,6 mm, furent créées autour des troisièmes et quatrièmes prémolaires chez 4 Labrador adultes. Chaque quadrant a été traité par des éponges de collagène résorbables utilisé comme vecteur (ASC) contenant rhBMP-2 (0.4 mg). Les quadrants contralatéraux des animaux furent aléatoirement distribués pour recevoir (ou pas) en plus un dispositif macro-poreux en ePTFE, en forme de dôme sur les implants de rhBMP-2/ACS. Les lambeaux furent déplacés pour recouvrir le dispositif en ePTFE et les dents et suturés. Les animaux furent sacrifiés après 8 semaines pour fournir des observations histologiques de la cicatrisation. Résultats: La cicatrisation ne posait pas de problèmes et on ne nota pas d'exposition des dispositifs. La moyenne de la formation osseuse était de (±SD) 4.7±0.2 mm (98%) et 4.5±0.4 mm (94%) de la hauteur de la lésion, respectivement, pour les quadrants ayant été traités par la rhBMP-2/ACS avec le dispositif en ePTFE ou la rhBMP-2/ACS seule (p>0.05). A l'inverse, la surface osseuse régénérée était significativement plus importante dans les quadrants traités par la rhBMP-2/ACS et les dispositifs en ePTFE par rapport au site traités seulement par la rhBMP-2/ACS (9.3±2.7 vs. 5.1±1.1 mm2; p<0.05). La formation cémentaire était similaire pour les deux groupes de traitement. L'ankylose compromettait la régénération parodontale dans tous les sites. Conclusions: Ces résultats suggèrent que le dispositif en ePTFE macro-poreux, qui assure un espace, semble convenir comme standard pour définir la formation osseuse induite par la rhBMP-2/ACS. [source] Gene expression analysis of immunostained endothelial cells isolated from formaldehyde-fixated paraffin embedded tumors using laser capture microdissection,A technical reportMICROSCOPY RESEARCH AND TECHNIQUE, Issue 12 2009Tomoatsu Kaneko Abstract Laser capture microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. In conventional LCM, frozen tissues stained with hematoxylin are normally used to the molecular analysis. Recent studies suggested that it is possible to carry out gene expression analysis of formaldehyde-fixated paraffin embedded (FFPE) tissues that were stained with hematoxylin. However, it is still unclear if quantitative gene expression analyses can be performed from LCM cells from FFPE tissues that were subjected to immunostaining to enhance identification of target cells. In this proof-of-principle study, we analyzed by reverse transcription-PCR (RT-PCR) and real time PCR the expression of genes in factor VIII immunostained human endothelial cells that were dissected from FFPE tissues by LCM. We observed that immunostaining should be performed at 4°C to preserve the mRNA from the cells. The expression of Bcl-2 in the endothelial cells was evaluated by RT-PCR and by real time PCR. Glyceraldehyde-3-phosphate dehydrogenase and 18S were used as house keeping genes for RT-PCR and real time PCR, respectively. This report unveils a method for quantitative gene expression analysis in cells that were identified by immunostaining and retrieved by LCM from FFPE tissues. This method is ideally suited for the analysis of relatively rare cell types within a tissue, and should improve on our ability to perform differential diagnosis of pathologies as compared to conventional LCM. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc. [source] A glimpse into the clinical proteome of human malaria parasites Plasmodium falciparum and Plasmodium vivaxPROTEOMICS - CLINICAL APPLICATIONS, Issue 11 2009Pragyan Acharya Abstract Malaria causes a worldwide annual mortality of about a million people. Rapidly evolving drug-resistant species of the parasite have created a pressing need for the identification of new drug targets and vaccine candidates. By developing fractionation protocols to enrich parasites from low-parasitemia patient samples, we have carried out the first ever proteomics analysis of clinical isolates of early stages of Plasmodium falciparum (Pf) and P. vivax. Patient-derived malarial parasites were directly processed and analyzed using shotgun proteomics approach using high-sensitivity MS for protein identification. Our study revealed about 100 parasite-coded gene products that included many known drug targets such as Pf hypoxanthine guanine phosphoribosyl transferase, Pf L -lactate dehydrogenase, and Plasmepsins. In addition, our study reports the expression of several parasite proteins in clinical ring stages that have never been reported in the ring stages of the laboratory-cultivated parasite strain. This proof-of-principle study represents a noteworthy step forward in our understanding of pathways elaborated by the parasite within the malaria patient and will pave the way towards identification of new drug and vaccine targets that can aid malaria therapy. [source] Cancer and the blood,brain barrier: ,Trojan horses' for courses?BRITISH JOURNAL OF PHARMACOLOGY, Issue 2 2008M Mazza The blood,brain barrier (BBB) limits the bioavailability of most bioactive molecules and drugs in the CNS, leaving clinicians with only a few options for pharmacotherapy. In this issue Regina et al. demonstrate that a ,Trojan horse' drug conjugate, acting as a substrate of a physiological BBB receptor that facilitates transcytosis, significantly improves drug transport into the CNS. Specifically, the low-density lipoprotein receptor-related protein (LRP) is used to carry a conjugate of paclitaxel and Angiopep-2, an aprotinin-derived peptide, across the BBB. Interestingly, in its conjugated form paclitaxel circumvents the efflux pumps at the BBB but still retains its activity against microtubules. Importantly, the authors were able to demonstrate improved therapeutic efficacy of this approach in orthotopic models of primary and metastatic brain cancer. This proof-of-principle study thus represents a milestone for drug delivery across the BBB but also a starting point for studies exploring wider applicability and potential limitations of the approach. British Journal of Pharmacology (2008) 155, 149,151; doi:fn1; published online 30 June 2008 [source] ADLOC: An Aptamer-Displacement Assay Based on Luminescent Oxygen ChannelingCHEMISTRY - A EUROPEAN JOURNAL, Issue 36 2010Dipl.-Chem. Abstract Functional nucleic acids, such as aptamers and allosteric ribozymes, can sense their ligands specifically, thereby undergoing structural alterations that can be converted into a detectable signal. The direct coupling of molecular recognition to signal generation enables the production of versatile reporters that can be applied as molecular probes for various purposes, including high-throughput screening. Here we describe an unprecedented type of a nucleic acid-based sensor system and show that it is amenable to high-throughput screening (HTS) applications. The approach detects the displacement of an aptamer from its bound protein partner by means of luminescent oxygen channeling. In a proof-of-principle study we demonstrate that the format is feasible for efficient identification of small drug-like molecules that bind to a protein target, in this case to the Sec7 domain of cytohesin. We extended the approach to a new cytohesin-specific single chain DNA aptamer, C10.41, which exhibits a similar binding behavior to cytohesins but has the advantage of being more stable and easier to synthesize and to modify than the RNA-aptamer M69. The results obtained with both aptamers indicate the general suitability of the aptamer-displacement assay based on luminescent oxygen channelling (ADLOC) for HTS. We also analyzed the potential for false positive hits and identified from a library of 18,000 drug-like small molecules two compounds as strong singlet-oxygen quenchers. With full automation and the use of commercially available plate readers, we estimate that the ADLOC-based assay described here could be used to screen at least 100,000 compounds per day. [source] Modified-release hydrocortisone for circadian therapy: a proof-of-principle study in dexamethasone-suppressed normal volunteersCLINICAL ENDOCRINOLOGY, Issue 1 2008J. Newell-Price Summary Background All existing long-term glucocorticoid replacement therapy is suboptimal as the normal nocturnal rise and waking morning peak of serum cortisol is not reproduced. Aim To test whether it is possible to reproduce the normal overnight rise and morning peak in serum cortisol using an oral delayed and sustained release preparation of hydrocortisone (Cortisolds). Subjects and methods Six healthy normal male volunteers attended on two occasions, in a single-dose, open-label, nonrandomized study. Endogenous cortisol secretion was suppressed by administration of dexamethasone. Cortisolds (formulation A or B) was administered at 2200 h on day 1. Blood samples for measurement of cortisol were taken from 2200 h every 30 min until 0700 h, then hourly until 2200 h on day 2. Fifteen body mass index (BMI)-matched control subjects had serum cortisol levels measured at 20-min intervals for 24 h. Serum cortisol profiles and pharmacokinetics after Cortisolds were compared with those in controls. Results Formulations A and B were associated with delayed drug release (by 2 h and 4 h, respectively), with median peak cortisol concentrations at 4·5 h (0245 h) and 10 h (0800 h), respectively, thereby reproducing the normal early morning rise in serum cortisol. Total cortisol exposure was not different from controls. Conclusions For the first time we have shown that it is possible to mimic the normal circadian rhythm of circulating cortisol with an oral modified-release formulation of hydrocortisone, providing the basis for development of physiological circadian replacement therapy in patients with adrenal insufficiency. [source] | ||||||||||