[Show abstract][Hide abstract] ABSTRACT: A somatic deletion at the proximal end of canine chromosome 27 (CFA27) was recently reported in 50% of malignant mammary tumors. This region harbours the tumor suppressor gene prefoldin subunit 5 (PFDN5) and the deletion correlated with a higher Ki-67 score. PFDN5 has been described to repress c-MYC and is, therefore, a candidate tumor-suppressor and cancer-driver gene in canine mammary cancer. Aim of this study was to confirm the recurrent deletion in a larger number of tumors.
Droplet digital PCR for PFDN5 was performed in DNA from 102 malignant, 40 benign mammary tumors/dysplasias, 11 non-neoplastic mammary tissues and each corresponding genomic DNA from leukocytes. The copy number of PFDN5 was normalized to a reference amplicon on canine chromosome 32 (CFA32). Z-scores were calculated, based on Gaussian distributed normalized PFDN5 copy numbers of the leukocyte DNA. Z-scores ≤ -3.0 in tissue were considered as being indicative of the PFDN5 deletion and called as such. The Ki-67 proliferation index was assessed in a subset of 79 tissue samples by immunohistochemistry.
The deletion was confirmed in 24% of all malignant tumors, detected in only 7.5% of the benign tumors and was not present in any normal mammary tissue sample. The subgroup of solid carcinomas (n = 9) showed the highest frequency of the deletion (67%) and those malignomas without microscopical high fraction of benign tissue (n = 71) had a 32% frequency (p<0.01 vs. benign samples). The Ki-67 score was found to be significantly higher (p<0.05) in the PFDN5-deleted group compared to malignant tumors without the deletion.
A somatic deletion of the PFDN5 gene is recurrently present in canine mammary cancer, supporting a potential role in carcinogenesis. The association of this deletion with higher Ki-67 indicates an increased proliferation rate and thus a link to tumor aggressiveness can be hypothesized. The confirmation of earlier results warrants further studies on PFDN5 as cancer-driver gene.
PLoS ONE 07/2015; 10(7):e0131280. DOI:10.1371/journal.pone.0131280 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Degradable implant material for bone remodeling that corresponds to the physiological stability of bone has still not been developed. Promising degradable materials with good mechanical properties are magnesium and magnesium alloys. However, excessive gas production due to corrosion can lower the biocompatibility. In the present study we used the polymer coating polycaprolactone (PCL), intended to lower the corrosion rate of magnesium. Additionally, improvement of implant geometry can increase bone remodeling. Porous structures are known to support vessel ingrowth and thus increase osseointegration. With the selective laser melting (SLM) process, defined open porous structures can be created. Recently, highly reactive magnesium has also been processed by SLM. We performed studies with a flat magnesium layer and with porous magnesium implants coated with polymers. The SLM produced magnesium was compared with the titanium alloy TiAl6V4, as titanium is already established for the SLM-process. For testing the biocompatibility, we used primary murine osteoblasts. Results showed a reduced corrosion rate and good biocompatibility of the SLM produced magnesium with PCL coating.
International Journal of Molecular Sciences 06/2015; 16(6):13287-13301. DOI:10.3390/ijms160613287 · 2.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Displacement of canine intervertebral disk material can be seen directly in diagnostic imaging modalities such as magnetic resonance imaging and computed tomographic imaging. Canine intervertebral disk herniation can be differentiated into Hansen type 1 and 2 categories by clinical appearance, but anular- and nuclear disk material cannot be distinguished in computed tomographic images. Therefore, we hypothesized that the "Disk extension beyond the interspace"-nomenclature that describes the displacement by the disk contour might aid diagnosis. The aim of this study was to test the reliability of the "Disk extension beyond the interspace"-nomenclature in the evaluation of canine intervertebral disks via magnetic resonance and computed tomographic imaging.
Magnetic resonance and computed tomographic images of 144 intervertebral disks of 43 dogs were evaluated by 3 observers with different degrees of experience from 2 institutions retrospectively. A substantial intraobserver agreement was found, while interobserver agreement was fair to moderate with significant differences in evaluation. Comparison of imaging methods showed a fair to moderate agreement without statistically significant differences in evaluation.
DEBIT-nomenclature cannot be recommended for veterinary clinical usage yet. The largest variability was found in the evaluation of the bulged canine intervertebral disk. The observers' experience and the imaging method influenced DEBIT- evaluation only slightly, while training and working at different institutions influenced DEBIT-evaluation strongly.
BMC Veterinary Research 05/2015; 11(1):110. DOI:10.1186/s12917-015-0421-x · 1.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Canine prostate cancer represents a spontaneous animal model for the human counterpart. Cells with stem cell-like character are considered to play a major role in therapeutic resistance and tumor relapse. Thus, the identification of markers allowing for recognition and characterization of these cells is essential. MATERIALS AND METHODS: Expression of 12 stem cell marker genes in the canine prostate cancer cell line CT1258 and spheroid cells generated from these was analyzed by quantitative real-time PCR. In CT1258 and the generated spheroid cells, CD44 and CD133 expression was analyzed by flow cytometry, as well as proliferation and doxorubicin resistance. RESULTS: Integrin alpha-6 (ITGA6) expression and metabolic activity were significantly up-regulated in CT1258-derived spheroid cells, while doxorubicin resistance remained comparable. CONCLUSION: ITGA6 de-regulation and metabolic activity appear to be characteristic of the generated spheres, indicating potential intervention targets.
Anticancer research 04/2015; 35(4):1917-27. · 1.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To improve well-known titanium implants, pores can be used for increasing bone formation and close bone-implant interface. Selective Laser Melting (SLM) enables the production of any geometry and was used for implant production with 250-µm pore size. OPEN ACCESS Int. J. Mol. Sci. 2015, 16 7479 The used pore size supports vessel ingrowth, as bone formation is strongly dependent on fast vascularization. Additionally, proangiogenic factors promote implant vascularization. To functionalize the titanium with proangiogenic factors, polycaprolactone (PCL) coating can be used. The following proangiogenic factors were examined: vascular endothelial growth factor (VEGF), high mobility group box 1 (HMGB1) and chemokine (C-X-C motif) ligand 12 (CXCL12). As different surfaces lead to different cell reactions, titanium and PCL coating were compared. The growing into the porous titanium structure of primary osteoblasts was examined by cross sections. Primary osteoblasts seeded on the different surfaces were compared using Live Cell Imaging (LCI). Cross sections showed cells had proliferated, but not migrated after seven days. Although the cell count was lower on titanium PCL implants in LCI, the cell count and cell spreading area development showed promising results for titanium PCL implants. HMGB1 showed the highest migration capacity for stimulating the endothelial cell line. Future perspective would be the incorporation of HMGB1 into PCL polymer for the realization of a slow factor release.
International Journal of Molecular Sciences 04/2015; 16(4):7478-7492. DOI:10.3390/ijms16047478 · 2.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
In molecular medicine, the manipulation of cells is prerequisite to evaluate genes as therapeutic targets or to transfect cells to develop cell therapeutic strategies. To achieve these purposes it is essential that given transfection techniques are capable of handling high cell numbers in reasonable time spans. To fulfill this demand, an alternative nanoparticle mediated laser transfection method is presented herein. The fs-laser excitation of cell-adhered gold nanoparticles evokes localized membrane permeabilization and enables an inflow of extracellular molecules into cells.ResultsThe parameters for an efficient and gentle cell manipulation are evaluated in detail. Efficiencies of 90% with a cell viability of 93% were achieved for siRNA transfection. The proof for a molecular medical approach is demonstrated by highly efficient knock down of the oncogene HMGA2 in a rapidly proliferating prostate carcinoma in vitro model using siRNA. Additionally, investigations concerning the initial perforation mechanism are conducted. Next to theoretical simulations, the laser induced effects are experimentally investigated by spectrometric and microscopic analysis. The results indicate that near field effects are the initial mechanism of membrane permeabilization.Conclusion
This methodical approach combined with an automated setup, allows a high throughput targeting of several 100,000 cells within seconds, providing an excellent tool for in vitro applications in molecular medicine. NIR fs lasers are characterized by specific advantages when compared to lasers employing longer (ps/ns) pulses in the visible regime. The NIR fs pulses generate low thermal impact while allowing high penetration depths into tissue. Therefore fs laser could be used for prospective in vivo applications.
Journal of Nanobiotechnology 02/2015; 13(1):10. DOI:10.1186/s12951-014-0057-1 · 4.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gold nanoparticle mediated (GNOME) laser transfection is a powerful technique to deliver small biologically relevant molecules into cells. However, the transfection of larger and especially negatively charged DNA remains challenging. The efficiency for pDNA was 0.57% using parameter that does not influence the endo- and exogenous DNA. In order to gain a deeper understanding of the actual molecule uptake process, the uptake efficiency was determined using molecules of different sizes. It was evaluated that uncharged dextran molecules (2000 kDa) were delivered with an efficiency of 68%. The intracellular distribution of injected molecules was visualized and larger molecules were primary found in the cytoplasm. Patch clamp measurements suggested a permeabilization time up to 15 minutes. The uptake efficiency depended on the size and charge of the molecule to deliver as well as the cell size. A minor role for transfection plays the cell type since primary stem cells were successfully transfected. The perforation efficiency of semi-adherent and suspension cells is influenced by the cell and molecule size.
Journal of Biophotonics 11/2014; 9999(9999). DOI:10.1002/jbio.201400065 · 3.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
Right ventricular (RV) volume and function are important diagnostic and prognostic factors in dogs with primary or secondary right-sided heart failure. The complex shape of the right ventricle and its retrosternal position make the quantification of its volume difficult. For that reason, only few studies exist, which deal with the determination of RV volume parameters. In human medicine cardiac magnetic resonance imaging (CMRI) is considered to be the reference technique for RV volumetric measurement (Nat Rev Cardiol 7(10):551-563, 2010), but cardiac computed tomography (CCT) and three-dimensional echocardiography (3DE) are other non-invasive methods feasible for RV volume quantification. The purpose of this study was the comparison of 3DE and CCT with CMRI, the gold standard for RV volumetric quantification.Results3DE showed significant lower and CCT significant higher right ventricular volumes than CMRI. Both techniques showed very good correlations (R¿>¿0.8) with CMRI for the volumetric parameters end-diastolic volume (EDV) and end-systolic volume (ESV). Ejection fraction (EF) and stroke volume (SV) were not different when considering CCT and CMRI, whereas 3DE showed a significant higher EF and lower SV than CMRI. The 3DE values showed excellent intra-observer variability (<3%) and still acceptable inter-observer variability (<13%).ConclusionCCT provides an accurate image quality of the right ventricle with comparable results to the reference method CMRI. CCT overestimates the RV volumes; therefore, it is not an interchangeable method, having the disadvantage as well of needing general anaesthesia. 3DE underestimated the RV-Volumes, which could be explained by the worse image resolution. The excellent correlation between the methods indicates a close relationship between 3DE and CMRI although not directly comparable. 3DE is a promising technique for RV volumetric quantification, but further studies in awake dogs and dogs with heart disease are necessary to evaluate its usefulness in veterinary cardiology.
BMC Veterinary Research 10/2014; 10(1):242. DOI:10.1186/s12917-014-0242-3 · 1.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objective: Although the classification of canine intervertebral disc degeneration using magnetic resonance imaging (MRI) has been described in the literature, there is no such classification using computed tomographic imaging. Because computed tomography (CT) is a frequently used diagnostic imaging tool in veterinary medicine, the aim of this study was the introduction and validation of such a scoring system. T2-weighted magnetic resonance images were available for comparative analysis. Material and methods: A total of 43 dogs were examined using CT and MRI. Image data records of 144 intervertebral discs were blinded, randomized and evaluated twice by three observers. CT data were analyzed using a self-developed scoring system, while MRI data sets were evaluated using the Pfirrmann scoring system. Intra- and interobserver agreement were determined using Statistical Analysis Software (SAS). Results: Intra- and interobserver agreement were mostly substantial in the Pfirrmann (0.58-0.77) and self-developed (0.60-0.81) scoring systems. A slight agreement was found between both classification systems (κ scores 0.26-0.29). Conclusion and clinical relevance: The self-developed scoring system allows a reliable assessment of canine intervertebral disc degeneration using CT imaging. Therefore, further diagnostic and prognostic information can be obtained. Degenerative changes in the intervertebral discs could be identified at an earlier stage when using MRI in comparison with CT.