Multipotency and self-renewal are considered as most important features of stem cells to persist throughout life in tissues. In this context, the role of HMGA proteins to influence proliferation of adipose-derived mesenchymal stem cell (ASCs) while maintaining their multipotent and self-renewal capacities has not yet been investigated. Therefore, extracellular HMGA1 and HMGA2 application alone (10-200 ng/mL) and in combination with each other (100, 200 ng/mL each) was investigated with regard to proliferative effects on canine ASCs (cASCs) after 48 hours of cultivation. Furthermore, mRNA expression of multipotency marker genes in unstimulated and HMGA2-stimulated cASCs (50, 100 ng/mL) was analyzed by RT-qPCR. HMGA1 significantly reduced cASCs proliferation in concentrations of 10-200 ng/mL culture medium. A combination of HMGA1 and HMGA2 protein (100 and 200 ng/mL each) caused the same effects, whereas no significant effect on cASCs proliferation was shown after HMGA2 protein application alone. RT-qPCR results showed that expression levels of marker genes including KLF4, SOX2, OCT4, HMGA2, and cMYC mRNAs were on the same level in both HMGA2-protein-stimulated and -unstimulated cASCs. Extracellular HMGA protein application might be valuable to control proliferation of cASCs in context with their employment in regenerative approaches without affecting their self-renewal and multipotency abilities.

UNLABELLED Background/Aim: Administration of stem cells is a promising novel approach for treatment of neurodegenerative diseases. For in vivo monitoring of transplanted cells, non-invasive imaging modalities are needed. In this study we determined the tracking efficiency of a superparamagnetic iron oxide (SPIO)-labelled canine cell line (MTH53A) in vitro as well as the human CD34(+) umbilical cord blood stem cells (hUCBCs) in vitro and in vivo efficiency by magnetic resonance imaging (MRI). MATERIALS AND METHODS SPIO-labelled MTH53A cells and hUCBCs were scanned in agar gel phantoms at 1.0 T or 7.0 T. For in vivo detection, 100,000 labelled hUCBCs were injected into the spinal cord of a transgenic amyotrophic lateral sclerosis (ALS) mouse and scanned at 7.0 T. RESULTS In vitro, 100,000 MTH53A cells and 250,000 hUCBCs were visible at 1.0 T. Scanning with 7.0 T revealed 25,000 detectable MTH53A cells. In vivo, 7.0 T MRI showed clear signals of 100,000 implanted cells. CONCLUSION MRI combined with SPIO nanoparticles provides valuable potential for non-invasive, non-toxic in vivo tracking of cells implanted into the spinal cord.

BACKGROUND: Mammary neoplasias are one of the most frequent and spontaneously occurring malignancies in dogs and humans. Due to the similar anatomy of the mammary gland in both species, the dog has become an important animal model for this cancer entity. In human breast carcinomas, the overexpression of a protein named high-mobility group box 1 (HMGB1) was reported. Cells of the immune system were described to release HMGB1 actively exerting cytokine function. Thereby it is involved in the immune system activation, tissue repair, and cell migration. Passive release of HMGB1 by necrotic cells at sites of tissue damage or in necrotic hypoxic regions of tumors induces cellular responses e.g. release of proinflammatory cytokines leading to elevated inflammatory response and neo-vascularization of necrotic tumor areas. Herein we investigated if a time-dependent stimulation with the separately applied proinflammatory cytokines TNF-α and IFN-γ can cause secretion of HMGB1 in a non-immune related HMGB1-non-secreting epithelial canine mammary cell line (MTH53A) derived from non-neoplastic tissue. METHODS: The canine cell line was transfected with recombinant HMGB1 bicistronic expression vectors and stimulated after transfection with the respective cytokine independently for 6, 24 and 48h. HMGB1 protein detection was performed by Western blot analysis and quantified a by enzyme-linked immunosorbent assay. Live cell laser scanning multiphoton microscopy of MTH53A cells expressing a HMGB1-GFP fusion protein was performed in order to examine, if secretion of HMGB1 under cytokine stimulating conditions is also visible by fluorescence imaging. RESULTS: The observed HMGB1 release kinetics showed a clearly time-dependent manner with a peak release 24h after TNF-α stimulation, while stimulation with IFN-γ had only small effects on the HMGB1 release. Multiphoton HMGB1 live cell microscopy showed diffuse cell membrane structure changes 29h after cytokine-stimulation but no clear secretion of HMGB1-GFP after TNF-α stimulation was visible. CONCLUSION: Our results demonstrate that non-immune HMGB1-non-secreting cells of epithelial origin derived from mammary non-neoplastic tissue can be induced to release HMGB1 by single cytokine application. This indicates that tumor and surrounding tissue can be stimulated by tumor present inflammatory and necrotic cytokines to release HMGB1 acting as neo-vascularizing factor thus promoting tumor growth.

BACKGROUND 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. RESULTS 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. CONCLUSIONS 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.

One of the main goals in cancer immunotherapy is the efficient activation of the host immune system against tumour cells. Dendritic cells (DCs) can induce specific anti-tumour immune responses in both experimental animal models and humans. However, most preclinical studies using small animal models show only limited correlation with studies carried out in clinical settings, whereas laboratory dogs naturally develop tumours that are biologically and histopathologically similar to their human counterparts. Here, we describe the generation and characterization of recombinant antibodies against canine DCs, isolated using the Tomlinson phage display system. We successfully isolated highly specific single-chain variable fragment (scFv) antibodies in a sequential three-step panning strategy involving depletion on canine peripheral blood mononuclear cells followed by positive selection on native canine DCs. This provides the basis for an antibody-based method for the immunological detection and manipulation of DCs and for monitoring antigen-specific immune responses.

Human and canine lymphoid neoplasms are characterized by non-random cytogenetic abnormalities. However, due to the low mitotic activity of the B cells, cytogenetic analyses of B-cell lymphoid proliferations are difficult to perform. In the present study we stimulated canine B-cell lymphoma cells with the immunostimulatory CpG-oligonucleotide DSP30 in combination with interleukin-2 (IL-2) and obtained an adequate number of metaphases. Cytogenetic analyses revealed the loss of one X chromosome as the sole cytogenetic aberration. Chromosome analysis of the corresponding blood showed a normal female karyotype. Monosomy X as the sole clonal chromosomal abnormality is found in human hematopoietic malignancies as well, thus the dog may serve as a promising animal model.

Besides man, the dog is the only known mammalian species that spontaneously develops carcinomas of the prostate with considerable frequency. For this reason, the dog is considered to be the only useful animal model for spontaneously occurring prostate malignancies in man. Cytogenetic investigations of human prostate cancers have revealed the frequent occurrence of trisomies 7, 8, and 17. Chromosome analyses of canine prostate carcinomas are rare. In this report we present 2 cases of canine prostate cancer showing a clonal polysomy 13 along with complex karyotype changes. Along with a previous report demonstrating polysomy 13 as the only karyotype deviation in a canine prostate cancer the present report supports the hypothesis that in canine prostate cancer, polysomy 13 is a recurrent cytogenetic aberration linked to the development of the disease. As human chromosomes (HSA) 8q and 4q and the canine chromosome (CFA) 13 share high homology, these results suggest that a conserved area on these chromosomes is involved in tumorigenesis in both species.

RAGE is a member of the immunoglobulin superfamily of cell surface molecules playing key roles in pathophysiological processes, e.g. immune/inflammatory disorders, Alzheimer's disease, diabetic arteriosclerosis and tumourigenesis. In humans 19 naturally occurring RAGE splicing variants resulting in either N-terminally or C-terminally truncated proteins were identified and are lately discussed as mechanisms for receptor regulation. Accordingly, deregulation of sRAGE levels has been associated with several diseases e.g. Alzheimer's disease, Type 1 diabetes, and rheumatoid arthritis. Administration of recombinant sRAGE to animal models of cancer blocked tumour growth successfully. In spite of its obvious relationship to cancer and metastasis data focusing sRAGE deregulation and tumours is rare. In this study we screened a set of tumours, healthy tissues and various cancer cell lines for RAGE splicing variants and analysed their structure. Additionally, we analysed the ratio of the mainly found transcript variants using quantitative Real-Time PCR. In total we characterised 24 previously not described canine and 4 human RAGE splicing variants, analysed their structure, classified their characteristics, and derived their respective protein forms. Interestingly, the healthy and the neoplastic tissue samples showed in majority RAGE transcripts coding for the complete receptor and transcripts showing insertions of intron 1.

ABSTRACT: BACKGROUND: Prostate cancer is a frequent finding in man. In dogs, malignant disease of the prostate is also of clinical relevance, although it is a less common diagnosis. Even though there are numerous differences in origin and development of the disease, man and dog share many similarities in the pathological presentation. For this reason, the dog might be a useful animal model for prostate malignancies in man. Although prostate cancer is of great importance in veterinary medicine as well as in comparative medicine, there are only few cell lines available. Thus, it was the aim of the present study to determine whether the formerly established prostate carcinoma cell line CT1258 is a suitable tool for in vivo testing, and to distinguish the growth pattern of the induced tumours. METHODS: For characterisation of the in vivo behaviour of the in vitro established canine prostate carcinoma cell line CT1258, cells were inoculated in 19 NOD.CB17-PrkdcScid/J (in the following: NOD-Scid) mice, either subcutaneously or intraperitoneally. After sacrifice, the obtained specimens were examined histologically and compared to the pattern of the original tumour in the donor. Cytogenetic investigation was performed. RESULTS: The cell line CT 1258 not only showed to be highly tumourigenic after subcutaneous as well as intraperitoneal inoculation, but also mimicked the behaviour of the original tumour. CONCLUSION: Tumours induced by inoculation of the cell line CT1258 resemble the situation in naturally occurring prostate carcinoma in the dog, and thus could be used as in vivo model for future studies.

ABSTRACT: BACKGROUND: The high mobility group A1 proteins (HMGA1a/HMGA1b) are highly conserved between mammalian species and widely described as participating in various cellular processes. By inducing DNA conformation changes the HMGA1 proteins indirectly influence the binding of various transcription factors and therefore effect the transcription regulation. In humans chromosomal aberrations affecting the HMGA1 gene locus on HSA 6p21 were described to be the cause for various benign mesenchymal tumours while high titres of HMGA1 proteins were shown to be associated with the neoplastic potential of various types of cancer. Interestingly, the absence of HMGA1 proteins was shown to cause insulin resistance and diabetes in humans and mice. Due to the various similarities in biology and presentation of human and canine cancers the dog has joined the common rodent animal model for therapeutic and preclinical studies. Accordingly, the canine genome was sequenced completely twice but unfortunately this could not solve the structure of canine HMGA1 gene. RESULTS: Herein we report the characterisation of the genomic structure of the canine HMGA1 gene consisting of 7 exons and 6 introns spanning in total 9524bp, the in vivo localisation of the HMGA1 protein to the nucleus, and a chromosomal assignment of the gene by FISH to CFA12q11. Additionally, we evaluated a described canine HMGA1 exon 6 SNP in 55 Dachshunds. CONCLUSIONS: The performed characterisations will make comparative analyses of aberrations affecting the human and canine gene and proteins possible, thereby providing a basis for revealing mechanisms involved in HMGA1 related pathogenesis in both species.