Molecular techniques that detect canine lymphoma cells by their clonal antigen receptor gene rearrangement play an increasing role for diagnosis as well as for monitoring minimal residual disease during and after cytostatic therapy. However, the methods currently available are time-consuming and/or cost-intensive thus impeding the use in clinical routine. The aim of the present study was to develop and evaluate a real-time polymerase chain reaction (PCR) with subsequent melting curve analysis (MCA) for the detection of clonally rearranged antigen receptor genes in dogs with B and T cell lymphoma on non formalin-fixed and paraffin-embedded lymph node samples.

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. 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.

Background: There are several numerical investigations on bone remodelling after total hip arthroplasty (THA) on the basis of the finite element analysis (FEA). For such computations certain boundary conditions have to be defined. The authors chose a maximum of three static load situations, usually taken from the gait cycle because this is the most frequent dynamic activity of a patient after THA.Materials and methods: The numerical study presented here investigates whether it is useful to consider only one static load situation of the gait cycle in the FE calculation of the bone remodelling. For this purpose, 5 different loading cases were examined in order to determine their influence on the change in the physiological load distribution within the femur and on the resulting strain-adaptive bone remodelling. First, four different static loading cases at 25%, 45%, 65% and 85% of the gait cycle, respectively, and then the whole gait cycle in a loading regime were examined in order to regard all the different loadings of the cycle in the simulation.Results: The computed evolution of the apparent bone density (ABD) and the calculated mass losses in the periprosthetic femur show that the simulation results are highly dependent on the chosen boundary conditions.Conclusion: These numerical investigations prove that a static load situation is insufficient for representing the whole gait cycle. This causes severe deviations in the FE calculation of the bone remodelling. However, accompanying clinical examinations are necessary to calibrate the bone adaptation law and thus to validate the FE calculations.

Relapse with drug-resistant disease is the main cause of death in MYCN-amplified neuroblastoma patients. MYCN-amplified neuroblastoma cells in vitro are characterized by a failure to arrest at the G₁-S checkpoint after irradiation- or drug-induced DNA damage. We show that several MYCN-amplified cell lines harbor additional chromosomal aberrations targeting p53 and/or pRB pathway components, including CDK4/CCND1/MDM2 amplifications, p16INK4A/p14ARF deletions or TP53 mutations. Cells with these additional aberrations undergo significantly lower levels of cell death after doxorubicin treatment compared with MYCN-amplified cells, with no additional mutations in these pathways. In MYCN-amplified cells CDK4 expression is elevated, increasing the competition between CDK4 and CDK2 for binding p21. This results in insufficient p21 to inhibit CDK2, leading to high CDK4 and CDK2 kinase activity upon doxorubicin treatment. CDK4 inhibition by siRNAs, selective small compounds or p19INK4D overexpression partly restored G₁-S arrest, delayed S-phase progression and reduced cell viability upon doxorubicin treatment. Our results suggest a specific function of p19INK4D, but not p16INK4A, in sensitizing MYCN-amplified cells with a functional p53 pathway to doxorubicin-induced cell death. In summary, the CDK4/cyclin D-pRB axis is altered in MYCN-amplified cells to evade a G₁-S arrest after doxorubicin-induced DNA damage. Additional chromosomal aberrations affecting the p53-p21 and CDK4-pRB axes compound the effects of MYCN on the G₁ checkpoint and reduce sensitivity to cell death after doxorubicin treatment. CDK4 inhibition partly restores G₁-S arrest and sensitizes cells to doxorubicin-mediated cell death in MYCN-amplified cells with an intact p53 pathway.

Prostate cancer is worldwide the sixth leading cause of cancer related death in men thus early detection and successful treatment are still of major interest. The commonly performed screening of the prostate-specific antigen (PSA) is controversially discussed, as in many patients the prostate-specific antigen levels are chronically elevated in the absence of cancer. Due to the unsatisfying efficiency of available prostate cancer screening markers and the current treatment outcome of the aggressive hormone refractory prostate cancer, the evaluation of novel molecular markers and targets is considered an issue of high importance. MicroRNAs are relatively stable in body fluids orchestrating simultaneously the expression of many genes. These molecules are currently discussed to bear a greater diagnostic potential than protein-coding genes, being additionally promising therapeutic drugs and/or targets. Herein we review the potential impact of the microRNAlet-7family on prostate cancer and show how deregulation of several of its target genes could influence the cellular equilibrium in the prostate gland, promoting cancer development as they do in a variety of other human malignant neoplasias.

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. The 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. 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 lasers could be used for prospective in vivo applications.