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1.  Characterization of nanoparticle mediated laser transfection by femtosecond laser pulses for applications in molecular medicine 
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.
PMCID: PMC4323028  PMID: 25645721
Laser transfection; Plasmonics; Nanoparticles; Permeabilization mechanisms; siRNA; Gene delivery
2.  Quantification of right ventricular volume in dogs: a comparative study between three-dimensional echocardiography and computed tomography with the reference method magnetic resonance imaging 
BMC Veterinary Research  2014;10(1):242.
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.
3DE 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%).
CCT 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.
PMCID: PMC4209227  PMID: 25306140
Right ventricular volume; Dog; Three-dimensional echocardiography; Magnetic resonance imaging; Computed tomography
3.  HMGA1 and HMGA2 expression and comparative analyses of HMGA2, Lin28 and let-7 miRNAs in oral squamous cell carcinoma 
BMC Cancer  2014;14(1):694.
Humans and dogs are affected by squamous cell carcinomas of the oral cavity (OSCC) in a considerably high frequency. The high mobility group A2 (HMGA2) protein was found to be highly expressed in human OSCC and its expression was suggested to act as a useful predictive and prognostic tool in clinical management of oral carcinomas. Herein the expression of HMGA2 and its sister gene HMGA1 were analysed within human and canine OSCC samples. Additionally, the HMGA negatively regulating miRNAs of the let-7 family as well as the let-7 regulating gene Lin28 were also comparatively analysed. Deregulations of either one of these members could affect the progression of human and canine OSCC.
Expression levels of HMGA1, HMGA2, Lin28, let-7a and mir-98 were analysed via relative qPCR in primary human and canine OSCC, thereof derived cell lines and non-neoplastic samples. Additionally, comparative HMGA2 protein expression was analysed by immunohistochemistry.
In both species, a significant up-regulation of the HMGA2 gene was found within the neoplastic samples while HMGA1 expression did not show significant deregulations. Comparative analyses showed down-regulation of mir-98 in human samples and up-regulation of let-7a and mir-98 in canine neoplastic samples. HMGA2 immunostainings showed higher intensities within the invasive front of the tumours than in the centre of the tumour in both species.
HMGA2 could potentially serve as tumour marker in both species while HMGA1 might play a minor role in OSCC progression. Comparative studies indicate an inverse correlation of HMGA2 and mir-98 expression in human samples whereas in dogs no such characteristic could be found.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-694) contains supplementary material, which is available to authorized users.
PMCID: PMC4190370  PMID: 25245141
Squamous cell carcinoma; HMGA1; HMGA2; let-7; mir-98; Lin28; Animal model; Dogs; Comparative oncology
4.  Role of miRNA Let-7 and Its Major Targets in Prostate Cancer 
BioMed Research International  2014;2014:376326.
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 microRNA let-7 family 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.
PMCID: PMC4168040  PMID: 25276782
5.  Evaluation of a New Recombinant Oncolytic Vaccinia Virus Strain GLV-5b451 for Feline Mammary Carcinoma Therapy 
PLoS ONE  2014;9(8):e104337.
Virotherapy on the basis of oncolytic vaccinia virus (VACV) infection is a promising approach for cancer therapy. In this study we describe the establishment of a new preclinical model of feline mammary carcinoma (FMC) using a recently established cancer cell line, DT09/06. In addition, we evaluated a recombinant vaccinia virus strain, GLV-5b451, expressing the anti-vascular endothelial growth factor (VEGF) single-chain antibody (scAb) GLAF-2 as an oncolytic agent against FMC. Cell culture data demonstrate that GLV-5b451 virus efficiently infected, replicated in and destroyed DT09/06 cancer cells. In the selected xenografts of FMC, a single systemic administration of GLV-5b451 led to significant inhibition of tumor growth in comparison to untreated tumor-bearing mice. Furthermore, tumor-specific virus infection led to overproduction of functional scAb GLAF-2, which caused drastic reduction of intratumoral VEGF levels and inhibition of angiogenesis.
In summary, here we have shown, for the first time, that the vaccinia virus strains and especially GLV-5b451 have great potential for effective treatment of FMC in animal model.
PMCID: PMC4122492  PMID: 25093734
6.  Generation and Characterisation of a Canine EGFP-HMGA2 Prostate Cancer In Vitro Model 
PLoS ONE  2014;9(6):e98788.
The architectural transcription factor HMGA2 is abundantly expressed during embryonic development. In several malignant neoplasias including prostate cancer, high re-expression of HMGA2 is correlated with malignancy and poor prognosis. The let-7 miRNA family is described to regulate HMGA2 negatively. The balance of let-7 and HMGA2 is discussed to play a major role in tumour aetiology. To further analyse the role of HMGA2 in prostate cancer a stable and highly reproducible in vitro model system is precondition. Herein we established a canine CT1258-EGFP-HMGA2 prostate cancer cell line stably overexpressing HMGA2 linked to EGFP and in addition the reference cell line CT1258-EGFP expressing solely EGFP to exclude EGFP-induced effects. Both recombinant cell lines were characterised by fluorescence microscopy, flow cytometry and immunocytochemistry. The proliferative effect of ectopically overexpressed HMGA2 was determined via BrdU assays. Comparative karyotyping of the derived and the initial CT1258 cell lines was performed to analyse chromosome consistency. The impact of the ectopic HMGA2 expression on its regulator let-7a was analysed by quantitative real-time PCR. Fluorescence microscopy and immunocytochemistry detected successful expression of the EGFP-HMGA2 fusion protein exclusively accumulating in the nucleus. Gene expression analyses confirmed HMGA2 overexpression in CT1258-EGFP-HMGA2 in comparison to CT1258-EGFP and native cells. Significantly higher let-7a expression levels were found in CT1258-EGFP-HMGA2 and CT1258-EGFP. The BrdU assays detected an increased proliferation of CT1258-HMGA2-EGFP cells compared to CT1258-EGFP and native CT1258. The cytogenetic analyses of CT1258-EGFP and CT1258-EGFP-HMGA2 resulted in a comparable hyperdiploid karyotype as described for native CT1258 cells. To further investigate the impact of recombinant overexpressed HMGA2 on CT1258 cells, other selected targets described to underlie HMGA2 regulation were screened in addition. The new fluorescent CT1258-EGFP-HMGA2 cell line is a stable tool enabling in vitro and in vivo analyses of the HMGA2-mediated effects on cells and the development and pathogenesis of prostate cancer.
PMCID: PMC4051699  PMID: 24914948
7.  CDK4 inhibition restores G₁-S arrest in MYCN-amplified neuroblastoma cells in the context of doxorubicin-induced DNA damage 
Cell Cycle  2013;12(7):1091-1104.
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.
PMCID: PMC3646865  PMID: 23462184
oncogene; RB1; TP53; cell cycle arrest; apoptosis
8.  Detection of clonal antigen receptor gene rearrangement in dogs with lymphoma by real-time polymerase chain reaction and melting curve analysis 
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.
In lymph node aspirates from 30 dogs with multicentric B cell lymphoma, real-time PCR with MCA detected clonal rearrangement in 100% and conventional PCR with polyacrylamide gel electrophoresis (PAGE) in 93% of samples. Both methods correctly identified clonality in 80% of lymph node aspirates of 10 dogs with T cell lymphoma. None of the two PCR systems detected clonal rearrangement in samples from 9 dogs with lymph node hyperplasia. Using a dilutional series with regular lymphoid desoxyribonucleic acid (DNA), detection limits of lymphoma DNA were as low as 0.8% and 6.25% for B and T cell clonal rearrangement with real-time PCR and MCA and at 3.13% and 12.5% with the conventional system. Median absolute detection limits of lymphoma DNA were shown to be at 0.1 ng and 1 ng for the B and T cell immunophenotype with the real-time PCR system and at 10 ng each with conventional PCR and PAGE.
Real-time PCR with MCA is a convenient and reliable method with a good analytical sensitivity. Thus, the method may assist the detection of clonal antigen receptor gene rearrangement in canine lymphoma patients in a clinical setting also in the presence of small amounts of neoplastic cells.
PMCID: PMC3904468  PMID: 24383544
Lymphoma; Dog; Real-time polymerase chain reaction; Melting curve analysis
9.  Adaptations in Muscle Activity to Induced, Short-Term Hindlimb Lameness in Trotting Dogs 
PLoS ONE  2013;8(11):e80987.
Muscle tissue has a great intrinsic adaptability to changing functional demands. Triggering more gradual responses such as tissue growth, the immediate responses to altered loading conditions involve changes in the activity. Because the reduction in a limb’s function is associated with marked deviations in the gait pattern, understanding the muscular responses in laming animals will provide further insight into their compensatory mechanisms as well as help to improve treatment options to prevent musculoskeletal sequelae in chronic patients. Therefore, this study evaluated the changes in muscle activity in adaptation to a moderate, short-term, weight-bearing hindlimb lameness in two leg and one back muscle using surface electromyography (SEMG). In eight sound adult dogs that trotted on an instrumented treadmill, bilateral, bipolar recordings of the m. triceps brachii, the m. vastus lateralis and the m. longissimus dorsi were obtained before and after lameness was induced. Consistent with the unchanged vertical forces as well as temporal parameters, neither the timing nor the level of activity changed significantly in the m. triceps brachii. In the ipsilateral m. vastus lateralis, peak activity and integrated SEMG area were decreased, while they were significantly increased in the contralateral hindlimb. In both sides, the duration of the muscle activity was significantly longer due to a delayed offset. These observations are in accordance with previously described kinetic and kinematic changes as well as changes in muscle mass. Adaptations in the activity of the m. longissimus dorsi concerned primarily the unilateral activity and are discussed regarding known alterations in trunk and limb motions.
PMCID: PMC3827467  PMID: 24236207
10.  Functional magnetic resonance imaging of the ascending stages of the auditory system in dogs 
Functional magnetic resonance imaging (fMRI) is a technique able to localize neural activity in the brain by detecting associated changes in blood flow. It is an essential tool for studying human functional neuroanatomy including the auditory system. There are only a few studies, however, using fMRI to study canine brain functions. In the current study ten anesthetized dogs were scanned during auditory stimulation. Two functional sequences, each in combination with a suitable stimulation paradigm, were used in each subject. Sequence 1 provided periods of silence during which acoustic stimuli could be presented unmasked by scanner noise (sparse temporal sampling) whereas in sequence 2 the scanner noise was present throughout the entire session (continuous imaging). The results obtained with the two different functional sequences were compared.
This study shows that with the proper experimental setup it is possible to detect neural activity in the auditory system of dogs. In contrast to human fMRI studies the strongest activity was found in the subcortical parts of the auditory pathways. Especially sequence 1 showed a high reliability in detecting activated voxels in brain regions associated with the auditory system.
These results indicate that fMRI is applicable for studying the canine auditory system and could become an additional method for the clinical evaluation of the auditory function of dogs. Additionally, fMRI is an interesting technique for future studies concerned with canine functional neuroanatomy.
PMCID: PMC3854503  PMID: 24131784
fMRI; Dog; Auditory pathways; Anesthesia
11.  Ontogenetic allometry of the Beagle 
Mammalian juveniles undergo dramatic changes in body conformation during development. As one of the most common companion animals, the time line and trajectory of a dog’s development and its body’s re-proportioning is of particular scientific interest. Several ontogenetic studies have investigated the skeletal development in dogs, but none has paid heed to the scapula as a critical part of the mammalian forelimb. Its functional integration into the forelimb changed the correspondence between fore- and hindlimb segments and previous ontogenetic studies observed more similar growth patterns for functionally than serially homologous elements. In this study, the ontogenetic development of six Beagle siblings was monitored between 9 and 51 weeks of age to investigate their skeletal allometry and compare this with data from other lines, breeds and species.
Body mass increased exponentially with time; log linear increase was observed up to the age of 15 weeks. Compared with body mass, withers and pelvic height as well as the lengths of the trunk, scapula, brachium and antebrachium, femur and crus exhibited positive allometry. Trunk circumference and pes showed negative allometry in all, pelvis and manus in most dogs. Thus, the typical mammalian intralimb re-proportioning with the proximal limb elements exhibiting positive allometry and the very distal ones showing negative allometry was observed. Relative lengths of the antebrachium, femur and crus increased, while those of the distal elements decreased.
Beagles are fully-grown regarding body height but not body mass at about one year of age. Particular attention should be paid to feeding and physical exertion during the first 15 weeks when they grow more intensively. Compared with its siblings, a puppy’s size at 9 weeks is a good indicator for its final size. Among siblings, growth duration may vary substantially and appears not to be related to the adult size. Within breeds, a longer time to physically mature is hypothesized for larger-bodied breeding lines. Similar to other mammals, the Beagle displayed nearly optimal intralimb proportions throughout development. Neither the forelimbs nor the hindlimbs conformed with the previously observed proximo-distal order of the limb segment’s growth gradients. Potential factors responsible for variations in the ontogenetic allometry of mammals need further evaluation.
PMCID: PMC4124762  PMID: 24112520
Scaling; Limb proportions; Body proportions; Bone growth; Serial homology; Body mass
12.  Microstructural analysis of pineal volume using trueFISP imaging 
World Journal of Radiology  2013;5(4):166-172.
AIM: To determine the spectrum of pineal microstructures (solid/cystic parts) in a large clinical population using a high-resolution 3D-T2-weighted sequence.
METHODS: A total of 347 patients enrolled for cranial magnetic resonance imaging were randomly included in this study. Written informed consent was obtained from all patients. The exclusion criteria were artifacts or mass lesions prohibiting evaluation of the pineal gland in any of the sequences. True-FISP-3D-imaging (1.5-T, isotropic voxel 0.9 mm) was performed in 347 adults (55.4 ± 18.1 years). Pineal gland volume (PGV), cystic volume, and parenchyma volume (cysts excluded) were measured manually.
RESULTS: Overall, 40.3% of pineal glands were cystic. The median PGV was 54.6 mm3 (78.33 ± 89.0 mm3), the median cystic volume was 5.4 mm3 (15.8 ± 37.2 mm3), and the median parenchyma volume was 53.6 mm3 (71.9 ± 66.7 mm3). In cystic glands, the standard deviation of the PGV was substantially higher than in solid glands (98% vs 58% of the mean). PGV declined with age (r = -0.130, P = 0.016).
CONCLUSION: The high interindividual volume variation is mainly related to cysts. Pineal parenchyma volume decreased slightly with age, whereas gender-related effects appear to be negligible.
PMCID: PMC3647208  PMID: 23671752
Pineal gland volume; Pineal cyst; Magnetic resonance imaging; Etiology; Reference range
13.  Comparison of non-coding RNAs in human and canine cancer 
The discovery of the post-transcriptional gene silencing (PTGS) by small non-protein-coding RNAs is considered as a major breakthrough in biology. In the last decade we just started to realize the biologic function and complexity of gene regulation by small non-coding RNAs. PTGS is a conserved phenomenon which was observed in various species such as fungi, worms, plants, and mammals. Micro RNAs (miRNA) and small interfering RNAs (siRNAs) are two gene silencing mediators constituting an evolutionary conserved class of non-coding RNAs regulating many biological processes in eukaryotes. As this small RNAs appear to regulate gene expression at translational and transcriptional level it is not surprising that during the last decade many human diseases among them Alzheimer's disease, cardiovascular diseases, and various cancer types were associated with deregulated miRNA expression. Consequently small RNAs are considered to hold big promises as therapeutic agents. However, despite of the enormous therapeutic potential many questions remain unanswered. A major critical point, when evaluating novel therapeutic approaches, is the transfer of in vitro settings to an in vivo model. Classical animal models rely on the laboratory kept animals under artificial conditions and often missing an intact immune system. Model organisms with spontaneously occurring tumors as e.g., dogs provide the possibility to evaluate therapeutic agents under the surveillance of an in intact immune system and thereby providing an authentic tumor reacting scenario. Considering the genomic similarity between canines and humans and the advantages of the dog as cancer model system for human neoplasias the analyses of the complex role of small RNAs in canine tumor development could be of major value for both species. Herein we discuss comparatively the role of miRNAs in human and canine cancer development and highlight the potential and advantages of the model organism dog for tumor research.
PMCID: PMC3619122  PMID: 23579348
RNAi; PTGS; model organism; cancer research; dog; miRNA; siRNA
14.  Diagnostic accuracy of a short-duration 3 Tesla magnetic resonance protocol for diagnosing stifle joint lesions in dogs with non-traumatic cranial cruciate ligament rupture 
Magnetic resonance (MR) imaging is the preferred diagnostic tool to evaluate internal disorders of many joints in humans; however, the usefulness of MR imaging in the context of osteoarthritis, and joint disease in general, has yet to be characterized in veterinary medicine. The objective of this study was to assess the diagnostic accuracy of short-duration 3 Tesla MR imaging for the evaluation of cranial and caudal cruciate ligament, meniscal and cartilage damage, as well as the degree of osteoarthritis, in dogs affected by non-traumatic, naturally-occurring cranial cruciate ligament rupture (CCLR). Diagnoses made from MR images were compared to those made during surgical exploration. Twenty-one client-owned dogs were included in this study, and one experienced evaluator assessed all images.
All cranial cruciate ligaments were correctly identified as ruptured. With one exception, all caudal cruciate ligaments were correctly identified as intact. High sensitivities and specificities were obtained when diagnosing meniscal rupture. MR images revealed additional subclinical lesions in both the cranial and caudal cruciate ligaments and in the menisci. There was a “clear” statistical (kappa) agreement between the MR and the surgical findings for both cartilage damage and degree of osteoarthritis. However, the large 95% confidence intervals indicated that evaluation of cartilage damage and of degree of osteoarthritis is not clinically satisfactory.
The presence of cruciate ligament damage and meniscal tears could be accurately assessed using the MR images obtained with our protocol. However, in the case of meniscal evaluation, occasional misdiagnosis did occur. The presence of cartilage damage and the degree of osteoarthritis could not be properly evaluated.
PMCID: PMC3599255  PMID: 23448526
Dog; Stifle; Cranial cruciate ligament; High-field MRI; Radiography
15.  Fore-Aft Ground Force Adaptations to Induced Forelimb Lameness in Walking and Trotting Dogs 
PLoS ONE  2012;7(12):e52202.
Animals alter their locomotor mechanics to adapt to a loss of limb function. To better understand their compensatory mechanisms, this study evaluated the changes in the fore-aft ground forces to forelimb lameness and tested the hypothesis that dogs unload the affected limb by producing a nose-up pitching moment via the exertion of a net-propulsive force when the lame limb is on the ground. Seven healthy Beagles walked and trotted at steady speed on an instrumented treadmill while horizontal force data were collected before and after a moderate lameness was induced. Peak, mean and summed braking and propulsive forces as well as the duration each force was exerted and the time to reach maximum force were evaluated for both the sound and the lame condition. Compared with the sound condition, a net-propulsive force was produced by the lame diagonal limbs due to a reduced braking force in the affected forelimb and an increased propulsive force in the contralateral hindlimb when the dogs walked and trotted. To regain pitch stability and ensure steady speed for a given locomotor cycle, the dogs produced a net-braking force when the sound diagonal limbs were on the ground by exerting greater braking forces in both limbs during walking and additionally reducing the propulsive force in the hindlimb during trotting. Consistent with the proposed mechanism, dogs maximize their double support phases when walking. Likely associated with the fore-aft force adaptations to lameness are changes in muscle recruitment that potentially result in short- and long-term effects on the limb and trunk muscles.
PMCID: PMC3530583  PMID: 23300614
16.  Virotherapy of Canine Tumors with Oncolytic Vaccinia Virus GLV-1h109 Expressing an Anti-VEGF Single-Chain Antibody 
PLoS ONE  2012;7(10):e47472.
Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for cancer therapy. We have previously reported that oncolytic vaccinia virus strains expressing an anti-VEGF (Vascular Endothelial Growth Factor) single-chain antibody (scAb) GLAF-1 exhibited significant therapeutic efficacy for treatment of human tumor xenografts. Here, we describe the use of oncolytic vaccinia virus GLV-1h109 encoding GLAF-1 for canine cancer therapy. In this study we analyzed the virus-mediated delivery and production of scAb GLAF-1 and the oncolytic and immunological effects of the GLV-1h109 vaccinia virus strain against canine soft tissue sarcoma and canine prostate carcinoma in xenograft models. Cell culture data demonstrated that the GLV-1h109 virus efficiently infect, replicate in and destroy both tested canine cancer cell lines. In addition, successful expression of GLAF-1 was demonstrated in virus-infected canine cancer cells and the antibody specifically recognized canine VEGF. In two different xenograft models, the systemic administration of the GLV-1h109 virus was found to be safe and led to anti-tumor and immunological effects resulting in the significant reduction of tumor growth in comparison to untreated control mice. Furthermore, tumor-specific virus infection led to a continued production of functional scAb GLAF-1, resulting in inhibition of angiogenesis. Overall, the GLV-1h109-mediated cancer therapy and production of immunotherapeutic anti-VEGF scAb may open the way for combination therapy concept i.e. vaccinia virus mediated oncolysis and intratumoral production of therapeutic drugs in canine cancer patients.
PMCID: PMC3473019  PMID: 23091626
17.  Longitudinal MRI contrast enhanced monitoring of early tumour development with manganese chloride (MnCl2) and superparamagnetic iron oxide nanoparticles (SPIOs) in a CT1258 based in vivo model of prostate cancer 
BMC Cancer  2012;12:284.
Cell lines represent a key tool in cancer research allowing the generation of neoplasias which resemble initial tumours in in-vivo animal models. The characterisation of early tumour development is of major interest in order to evaluate the efficacy of therapeutic agents. Magnetic resonance imaging (MRI) based in-vivo characterisation allows visualisation and characterisation of tumour development in early stages prior to manual palpation. Contrast agents for MRI such as superparamagnetic iron oxide nanoparticles (SPIOs) and manganese chloride (MnCl2) represent powerful tools for the in-vivo characterisation of early stage tumours. In this experimental study, we labelled prostate cancer cells with MnCl2 or SPIOs in vitro and used 1 T MRI for tracing labelled cells in-vitro and 7 T MRI for tracking in an in-vivo animal model.
Labelling of prostate cancer cells CT1258 was established in-vitro with MnCl2 and SPIOs. In-vitro detection of labelled cells in an agar phantom was carried out through 1 T MRI while in-vivo detection was performed using 7 T MRI after subcutaneous (s.c.) injection of labelled cells into NOD-Scid mice (n = 20). The animals were scanned in regular intervals until euthanization. The respective tumour volumes were analysed and corresponding tumour masses were subjected to histologic examination.
MnCl2in-vitro labelling resulted in no significant metabolic effects on proliferation and cell vitality. In-vitro detection-limit accounted 105 cells for MnCl2 as well as for SPIOs labelling. In-vivo 7 T MRI scans allowed detection of 103 and 104 cells. In-vivo MnCl2 labelled cells were detectable from days 4–16 while SPIO labelling allowed detection until 4 days after s.c. injection. MnCl2 labelled cells were highly tumourigenic in NOD-Scid mice and the tumour volume development was characterised in a time dependent manner. The amount of injected cells correlated with tumour size development and disease progression. Histological analysis of the induced tumour masses demonstrated characteristic morphologies of prostate adenocarcinoma.
To the best of our knowledge, this is the first study reporting direct in-vitro MnCl2 labelling and 7 T based in-vivo MRI tracing of cancer cells in a model of prostate cancer. MnCl2 labelling was found to be suitable for in-vivo tracing allowing long detection periods. The labelled cells kept their highly tumourigenic potential in-vivo. Tumour volume development was visualised prior to manual palpation allowing tumour characterisation in early stages of the disease.
PMCID: PMC3520113  PMID: 22784304
18.  Authentication of Primordial Characteristics of the CLBL-1 Cell Line Prove the Integrity of a Canine B-Cell Lymphoma in a Murine In Vivo Model 
PLoS ONE  2012;7(6):e40078.
Cell lines are key tools in cancer research allowing the generation of neoplasias in animal models resembling the initial tumours able to mimic the original neoplasias closely in vivo. Canine lymphoma is the major hematopoietic malignancy in dogs and considered as a valuable spontaneous large animal model for human Non-Hodgkin's Lymphoma (NHL). Herein we describe the establishment and characterisation of an in vivo model using the canine B-cell lymphoma cell line CLBL-1 analysing the stability of the induced tumours and the ability to resemble the original material. CLBL-1 was injected into Rag2−/−γc−/− mice. The generated tumor material was analysed by immunophenotyping and histopathology and used to establish the cell line CLBL-1M. Both cell lines were karyotyped for detection of chromosomal aberrations. Additionally, CLBL-1 was stimulated with IL-2 and DSP30 as described for primary canine B-cell lymphomas and NHL to examine the stimulatory effect on cell proliferation. CLBL-1 in vivo application resulted in lymphoma-like disease and tumor formation. Immunophenotypic analysis of tumorous material showed expression of CD45+, MHCII+, CD11a+ and CD79αcy+. PARR analysis showed positivity for IgH indicating a monoclonal character. These cytogenetic, molecular, immunophenotypical and histological characterisations of the in vivo model reveal that the induced tumours and thereof generated cell line resemble closely the original material. After DSP30 and IL-2 stimulation, CLBL-1 showed to respond in the same way as primary material. The herein described CLBL-1 in vivo model provides a highly stable tool for B-cell lymphoma research in veterinary and human medicine allowing various further in vivo studies.
PMCID: PMC3386195  PMID: 22761949
20.  Oncolytic virotherapy in veterinary medicine: current status and future prospects for canine patients 
Oncolytic viruses refer to those that are able to eliminate malignancies by direct targeting and lysis of cancer cells, leaving non-cancerous tissues unharmed. Several oncolytic viruses including adenovirus strains, canine distemper virus and vaccinia virus strains have been used for canine cancer therapy in preclinical studies. However, in contrast to human studies, clinical trials with oncolytic viruses for canine cancer patients have not been reported. An 'ideal' virus has yet to be identified. This review is focused on the prospective use of oncolytic viruses in the treatment of canine tumors - a knowledge that will undoubtedly contribute to the development of oncolytic viral agents for canine cancer therapy in the future.
PMCID: PMC3398296  PMID: 22216938
cancer; canine cancer therapy; oncolytic virus; oncolysis; target molecule, combination therapy
21.  Immunohistochemical investigation of Foxp3 expression in the intestine in healthy and diseased dogs 
Veterinary Research  2012;43(1):23.
Intestinal immune regulation including development of oral tolerance is of great importance for the maintenance of intestinal homeostasis. Concerning this, regulatory T cells (Tregs) occupy a pivotal role in cell-mediated immunosuppression. Dysregulation of mucosal immunology leading to an abnormal interaction with commensal bacteria is suggested to play a key role in the pathogenesis of Inflammatory Bowel Disease (IBD) in men and dogs. The aim of this study was to characterise the expression of Foxp3 in the normal canine gut of 18 dogs (mean age: 6.03 years), in 16 dogs suffering from IBD (mean age: 5.05 years), and of 6 dogs with intestinal nematode infection (mean age: 0.87 years) using immunohistochemistry. In the duodenum, Tregs in healthy dogs declined from villi (median: 10.67/62 500 μm2) to crypts (median: 1.89/62 500 μm2). Tregs were further increased in the villi of middle-aged dogs (median: 18.92/62 500 μm2) in contrast to juvenile (median: 3.50/62 500 μm2) and old (median: 9.56/62 500 μm2) individuals. Compared to healthy controls, animals suffering from IBD revealed reduced numbers of Tregs in duodenal villi (median: 4.13/62 500 μm2). Dogs with intestinal nematode infection displayed increased numbers of Tregs (median: 21.06/62 500 μm2) compared to healthy animals.
Age-related changes indicate a progressive establishment of oral tolerance and immunosenescence in the canine elderly. The results further suggest that a defect in Treg homeostasis may be involved in the pathogenesis of canine IBD. In contrast, increased numbers of Tregs in the duodenum may be due to nematode infection.
PMCID: PMC3364872  PMID: 22440243
22.  Effects of Pulse Phase Duration and Location of Stimulation Within the Inferior Colliculus on Auditory Cortical Evoked Potentials in a Guinea Pig Model 
The auditory midbrain implant (AMI), which consists of a single shank array designed for stimulation within the central nucleus of the inferior colliculus (ICC), has been developed for deaf patients who cannot benefit from a cochlear implant. Currently, performance levels in clinical trials for the AMI are far from those achieved by the cochlear implant and vary dramatically across patients, in part due to stimulation location effects. As an initial step towards improving the AMI, we investigated how stimulation of different regions along the isofrequency domain of the ICC as well as varying pulse phase durations and levels affected auditory cortical activity in anesthetized guinea pigs. This study was motivated by the need to determine in which region to implant the single shank array within a three-dimensional ICC structure and what stimulus parameters to use in patients. Our findings indicate that complex and unfavorable cortical activation properties are elicited by stimulation of caudal–dorsal ICC regions with the AMI array. Our results also confirm the existence of different functional regions along the isofrequency domain of the ICC (i.e., a caudal–dorsal and a rostral–ventral region), which has been traditionally unclassified. Based on our study as well as previous animal and human AMI findings, we may need to deliver more complex stimuli than currently used in the AMI patients to effectively activate the caudal ICC or ensure that the single shank AMI is only implanted into a rostral–ventral ICC region in future patients.
PMCID: PMC2975884  PMID: 20717834
auditory midbrain implant; auditory brainstem implant; cochlear implant; deep brain stimulation; auditory cortex; auditory thalamus
23.  Comparison of nanoparticle-mediated transfection methods for DNA expression plasmids: efficiency and cytotoxicity 
Reproducibly high transfection rates with low methodology-induced cytotoxic side effects are essential to attain the required effect on targeted cells when exogenous DNA is transfected. Different approaches and modifications such as the use of nanoparticles (NPs) are being evaluated to increase transfection efficiencies. Several studies have focused on the attained transfection efficiency after NP-mediated approaches. However, data comparing toxicity of these novel approaches with conventional methods is still rare.
Transfection efficiency and methodology-induced cytotoxicity were analysed after transfection with different NP-mediated and conventional approaches. Two eukaryotic DNA-expression-plasmids were used to transfect the mammalian cell line MTH53A applying six different transfection protocols: conventional transfection reagent (FuGENE HD, FHD), FHD in combination with two different sizes of stabilizer-free laser-generated AuNPs (PLAL-AuNPs_S1,_S2), FHD and commercially available AuNPs (Plano-AuNP), and two magnetic transfection protocols. 24 h post transfection efficiency of each protocol was analysed using fluorescence microscopy and GFP-based flow cytometry. Toxicity was assessed measuring cell proliferation and percentage of propidium iodide (PI%) positive cells. Expression of the respective recombinant proteins was evaluated by immunofluorescence.
The addition of AuNPs to the transfection protocols significantly increased transfection efficiency in the pIRES-hrGFPII-eIL-12 transfections (FHD: 16%; AuNPs mean: 28%), whereas the magnet-assisted protocols did not increase efficiency. Ligand-free PLAL-AuNPs had no significant cytotoxic effect, while the ligand-stabilized Plano-AuNPs induced a significant increase in the PI% and lower cell proliferation. For pIRES-hrGFPII-rHMGB1 transfections significantly higher transfection efficiency was observed with PLAL-AuNPs (FHD: 31%; PLAL-AuNPs_S1: 46%; PLAL-AuNPs_S2: 50%), while the magnet-assisted transfection led to significantly lower efficiencies than the FHD protocol. With PLAL-AuNPs_S1 and _S2 the PI% was significantly higher, yet no consistent effect of these NPs on cell proliferation was observed. The magnet-assisted protocols were least effective, but did result in the lowest cytotoxic effect.
This study demonstrated that transfection efficiency of DNA-expression-plasmids was significantly improved by the addition of AuNPs. In some combinations the respective cytotoxicity was increased depending on the type of the applied AuNPs and the transfected DNA construct. Consequently, our results indicate that for routine use of these AuNPs the specific nanoparticle formulation and DNA construct combination has to be considered.
PMCID: PMC3214154  PMID: 22014272
24.  Multidetector-row computed tomography of thoracic aortic anomalies in dogs and cats: Patent ductus arteriosus and vascular rings 
Diagnosis of extracardiac intrathoracic vascular anomalies is of clinical importance, but remains challenging. Traditional imaging modalities, such as radiography, echocardiography, and angiography, are inherently limited by the difficulties of a 2-dimensional approach to a 3-dimensional object. We postulated that accurate characterization of malformations of the aorta would benefit from 3-dimensional assessment. Therefore, multidetector-row computed tomography (MDCT) was chosen as a 3-dimensional, new, and noninvasive imaging technique. The purpose of this study was to evaluate patients with 2 common diseases of the intrathoracic aorta, either patent ductus arteriosus or vascular ring anomaly, by contrast-enhanced 64-row computed tomography.
Electrocardiography (ECG)-gated and thoracic nongated MDCT images were reviewed in identified cases of either a patent ductus arteriosus or vascular ring anomaly. Ductal size and morphology were determined in 6 dogs that underwent ECG-gated MDCT. Vascular ring anomalies were characterized in 7 dogs and 3 cats by ECG-gated MDCT or by a nongated thoracic standard protocol.
Cardiac ECG-gated MDCT clearly displayed the morphology, length, and caliber of the patent ductus arteriosus in 6 affected dogs. Persistent right aortic arch was identified in 10 animals, 8 of which showed a coexisting aberrant left subclavian artery. A mild dilation of the proximal portion of the aberrant subclavian artery near its origin of the aorta was present in 4 dogs, and a diverticulum analogous to the human Kommerell's diverticulum was present in 2 cats.
Contrast-enhanced MDCT imaging of thoracic anomalies gives valuable information about the exact aortic arch configuration. Furthermore, MDCT was able to characterize the vascular branching patterns in dogs and cats with a persistent right aortic arch and the morphology and size of the patent ductus arteriosus in affected dogs. This additional information can be of help with regard to improved diagnoses of thoracic anomalies and the planning of surgical interventions.
PMCID: PMC3186751  PMID: 21943366
25.  Significant Growth Inhibition of Canine Mammary Carcinoma Xenografts following Treatment with Oncolytic Vaccinia Virus GLV-1h68 
Journal of Oncology  2010;2010:736907.
Canine mammary carcinoma is a highly metastatic tumor that is poorly responsive to available treatment. Therefore, there is an urgent need to identify novel agents for therapy of this disease. Recently, we reported that the oncolytic vaccinia virus GLV-1h68 could be a useful tool for therapy of canine mammary adenoma in vivo. In this study we analyzed the therapeutic effect of GLV-1h68 against canine mammary carcinoma. Cell culture data demonstrated that GLV-1h68 efficiently infected and destroyed cells of the mammary carcinoma cell line MTH52c. Furthermore, after systemic administration, this attenuated vaccinia virus strain primarily replicated in canine tumor xenografts in nude mice. Finally, infection with GLV-1h68 led to strong inflammatory and oncolytic effects resulting in significant growth inhibition of the tumors. In summary, the data showed that the GLV-1h68 virus strain has promising potential for effective treatment of canine mammary carcinoma.
PMCID: PMC2902752  PMID: 20631910

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