Publications by authors named "Ingrid Walter"

70 Publications

Sexual Differentiation and Primordial Germ Cell Distribution in the Early Horse Fetus.

Animals (Basel) 2021 Aug 17;11(8). Epub 2021 Aug 17.

Center for Artificial Insemination and Embryo Transfer, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, Austria.

It was the aim of this study to characterize the development of the gonads and genital ducts in the equine fetus around the time of sexual differentiation. This included the identification and localization of the primordial germ cell population. Equine fetuses between 45 and 60 days of gestation were evaluated using a combination of micro-computed tomography scanning, immunohistochemistry, and multiplex immunofluorescence. Fetal gonads increased in size 23-fold from 45 to 60 days of gestation, and an even greater increase was observed in the metanephros volume. Signs of mesonephros atrophy were detected during this time. Tubular structures of the fetal testes were present from day 50 onwards, whereas cell clusters dominated in the fetal ovary. The genital ducts were well-differentiated and presented a lumen in all samples. No sign of mesonephric or paramesonephric duct degeneration was detected. Expression of AMH was strong in the fetal testes but absent in ovaries. Irrespective of sex, primordial germ cells selectively expressed LIN28. Migration of primordial germ cells from the mesonephros to the gonad was detected at 45 days, but not at 60 days of development. Their number and distribution within the gonad were influenced ( < 0.05) by fetal sex. Most primordial germ cells (86.8 ± 3.2% in females and 84.6 ± 4.7% in males) were characterized as pluripotent according to co-localization with CD117. However, only a very small percentage of primordial germ cells were proliferating (7.5 ± 1.7% in females and 3.2 ± 1.2% in males) based on co-localization with Ki67. It can be concluded that gonadal sexual differentiation in the horse occurs asynchronously with regard to sex but already before 45 days of gestation.
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http://dx.doi.org/10.3390/ani11082422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8388682PMC
August 2021

S-nitroso human serum albumin as a nitric oxide donor in drug-eluting vascular grafts: Biofunctionality and preclinical evaluation.

Acta Biomater 2021 Jul 28. Epub 2021 Jul 28.

Center for Biomedical Research, Medical University Vienna, Austria; Ludwig Boltzmann Institute for Cardiovascular Research, Vienna, Austria; Austrian Cluster for Tissue Regeneration, Vienna, Austria. Electronic address:

Currently available synthetic small diameter vascular grafts reveal low patency rates due to thrombosis and intimal hyperplasia. Biofunctionalized grafts releasing nitric oxide (NO) in situ may overcome these limitations. In this study, a drug-eluting vascular graft was designed by blending polycaprolactone (PCL) with S-nitroso-human-serum-albumin (S-NO-HSA), a nitric oxide donor with prolonged half-life. PCL-S-NO-HSA grafts and patches were fabricated via electrospinning. The fabrication process was optimized. Patches were characterized in vitro for their morphology, drug release, biomechanics, inflammatory effects, cell proliferation, and expression of adhesion molecules. The selected optimized formulation (8%PCL-S-NO-HSA) had superior mechanical/morphological properties with high protein content revealing extended NO release (for 28 days). 8%PCL-S-NO-HSA patches significantly promoted endothelial cell proliferation while limiting smooth muscle cell proliferation. Expression of adhesion molecules (ICAM-1, VCAM-1) and pro-inflammatory macrophage/cytokine markers (CD80, IL-1α, TNF-α) was significantly reduced. 8%PCL-S-NO-HSA patches had superior immunomodulatory properties by up-regulating anti-inflammatory cytokines (IL-10) and M2 macrophage marker (CD163) at final time points. Grafts were further evaluated in a small rodent model as aortic implants up to 12 weeks. Grafts were assessed by magnetic resonance imaging angiography (MRI) in vivo and after retrieval by histology. All grafts remained 100 % patent with no signs of thrombosis or calcification. 8%PCL-S-NO-HSA vascular grafts supported rapid endothelialization, whereas smooth muscle cell proliferation was hampered in earlier phases. This study indicates that 8%PCL-S-NO-HSA grafts effectively support long-term in situ release of bioactive NO. The beneficial effects observed can be promising features for long-term success of small diameter vascular grafts. STATEMENT OF SIGNIFICANCE: Despite extensive research in the field of small diameter vascular graft replacement, there is still no appropriate substitute to autografts yet. Various limitations are associated with currently available synthetic vascular grafts such as thrombogenicity and intimal hyperplasia. Therefore, developing new generations of such conduits has become a major focus of research. One of the most significant signaling molecules that are involved in homeostasis of the vascular system is nitric oxide. The new designed nitric-oxide eluting vascular grafts described in this study induce rapid surface endothelialization and late migration of SMCs into the graft wall. These beneficial effects have potential to improve current limitations of small diameter vascular grafts.
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http://dx.doi.org/10.1016/j.actbio.2021.07.048DOI Listing
July 2021

Effect of prednisolone pre-treatment on cat lymphoma cell sensitivity towards chemotherapeutic drugs.

Res Vet Sci 2021 Sep 15;138:178-187. Epub 2021 Jun 15.

Clinic for Companion Animal Medicine, Unit for Internal Medicine, University of Veterinary Medicine, Vienna, Austria.

Corticosteroid administration prior to the application of chemotherapy in small animal lymphoma patients is a concern, as it is discussed to negatively influence the therapeutic outcome due to corticosteroid-induced drug resistance. Using feline lymphoma cell lines FT-1 and MS4 we have shown, that prednisolone pre-treatment alters the susceptibility of these cells towards doxorubicin or vincristine treatment in vitro. The observed effect was negative as for the killing potential and it was cell line and drug (doxorubicin or vincristine) dependent. Furthermore, increase in mRNA expression of selected proteins with multidrug resistance potential (MDR1, BCRP, LRP, MT) was observed after prednisolone pre-treatment. Administration of chemical inhibitors of these proteins did not lead to reversal in sensitivity of tested cell lines to doxorubicin or vincristine.
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http://dx.doi.org/10.1016/j.rvsc.2021.06.014DOI Listing
September 2021

Upregulation of signal transducer and activator of transcription 3 in dogs with chronic inflammatory enteropathies.

J Vet Intern Med 2021 May 6;35(3):1288-1296. Epub 2021 May 6.

Internal Medicine, Clinic for Small Animals, Department for Companion Animals and Horses, University of Veterinary Medicine, Vienna, Austria.

Background: In inflammatory bowel disease (IBD) in humans, phosphorylated signal transducer and activator of transcription 3 (pSTAT3) is upregulated in mucosal epithelial cells and correlates with clinical severity.

Hypothesis/objective: To investigate the expression pattern of pSTAT3 in the mucosa of dogs with chronic inflammatory enteropathy (CIE) and explore correlations between its expression and clinical and histopathological severity scoring.

Animals: Twenty-eight canine CIE patients grouped into food-responsive enteropathy (FRE;  9), steroid-responsive enteropathy (SRE;  10), and protein-losing enteropathy (PLE;  9). Ten healthy beagle dogs served as controls (CO).

Methods: Retrospective case control study. Immunohistochemistry was used to detect pSTAT3 in canine duodenal mucosa samples.

Results: Compared to CO, SRE (P < .001) and PLE (P < .001) dogs had significantly higher pSTAT3 expression in the villus epithelium. The SRE group had a significantly higher expression in the villus lamina propria (VLP) compared to controls (P = .009). In the crypt epithelium (CE), all CIE dogs had significantly higher pSTAT3 expression (FRE, P = .002; SRE, P = .003; PLE, P < .001) compared to CO. In the lamina propria crypt region (CLP), dogs with FRE (P = .04) and SRE (P = .03) had significantly upregulated pSTAT3 compared to controls. A positive correlation was found between canine chronic enteropathy clinical activity index (CCECAI) scoring and pSTAT3 expression for both epithelial (rho = .541; P < .001) and crypt regions (rho = .32; P = .02).

Conclusions And Clinical Importance: pSTAT3 is upregulated in CIE in dogs, correlates with clinical severity, and may be helpful as a clinical marker in dogs with CIE.
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http://dx.doi.org/10.1111/jvim.16141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8163116PMC
May 2021

Expression of Enzymes Associated with Prostaglandin Synthesis in Equine Conceptuses.

Animals (Basel) 2021 Apr 20;11(4). Epub 2021 Apr 20.

Platform for Artificial Insemination and Embryo Transfer, Department for Small Animals and Horses, Vetmeduni Vienna, Veterinärplatz 1, 1210 Vienna, Austria.

In the horse, mobility of the conceptus is required for maternal recognition of pregnancy depending on secretion of prostaglandins by the conceptus. The aim of this study was to determine the expression and localization of key enzymes of the different pathways leading to synthesis of prostaglandin E2 and F2α in the equine conceptus during the mobility phase. Enzyme expression was analyzed via quantitative RT-PCR in total RNA samples of equine conceptuses collected on days 10 (n = 5), 12 (n = 12), 14 (n = 5) and 16 (n = 7) from healthy mares. Relative abundance of cyclooxygenase ()-2 mRNA was higher ( < 0.05) than of -1 irrespective of conceptus age and for phospholipase A2 on day 16 in comparison to all other days ( < 0.01). Abundance of mRNA of cytosolic and microsomal prostaglandin E synthase () and of carbonyl reductase () 1 was not influenced by conceptus age. Immunohistochemically, COX-1, COX-2, as well as cytosolic and microsomal PGES were present in both the ectodermal and endodermal layer of the yolk sac wall. CBR-1 was restricted to periembryonic disc area. The localisation of the key enzymes explains the mechanism of embryo mobility. In vitro incubation of primary trophoblast cell cultures with oxytocin had no effect on key enzyme synthesis.
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http://dx.doi.org/10.3390/ani11041180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8074782PMC
April 2021

Expression of COX-2, p16, and Ki67 in the range from normal breast tissue to breast cancer.

Neoplasma 2021 Mar 5;68(2):342-351. Epub 2020 Nov 5.

2nd Department of Gynecology and Obstetrics, Faculty of Medicine, Comenius University in Bratislava, Bratislava, Slovakia.

The increasing number of diagnosed breast lesions lead to the critical need for new markers that would elucidate the process of tumorigenesis. The objective of the study was to examine COX-2, p16, and Ki67 expression in a broad spectrum of breast lesions in order to define the proteins' phenotype throughout the tumorigenesis. Expression was studied by immunohistochemistry in 308 human breast samples divided into 7 subgroups - flat epithelial atypia (FEA), atypical hyperplasia (ADH), intraductal carcinoma (DCIS), invasive cancer (IC), benign lesions (BLs), normal tissue adjacent to breast cancer (CANT), and fatty tissue (FT). Analysis among 4 subgroups - premalignant lesions (DIN), IC, BLs, and normal tissue was also performed. High prevalence of COX-2 overexpression was found in all breast lesions including BLs (70% FEA, 89% ADH, 86% DCIS, 81% IC, 44% CANT, 92% BLs, 29% FT). Significant dominance of p16 overexpression was found in premalignant lesions and BLs (50% FEA, 67% ADH, 50% DCIS, 37% IC, 8% CANT, 58% BLs, 21% FT). The location of staining within p16+ cells differed - BLs showed nuclear positivity, whereas in IC it was exclusively cytoplasmic. Premalignant lesions showed all types of p16 positivity. Significantly higher prevalence of COX-2+p16+Ki67+ phenotype was in premalignant tumors with the highest prevalence in ADH (40% of FEA, 67% ADH, 35% DCIS, 20% IC, 3% CANT, 20% BLs, 14% FT). Our observations showed a high prevalence of COX-2+p16+Ki67+ phenotype in premalignant lesions. Further studies are needed in order to elucidate if this phenotype reflects any specific pathway of future progression of premalignant breast lesions.
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http://dx.doi.org/10.4149/neo_2020_200731N798DOI Listing
March 2021

Assessment of a long-term in vitro model to characterize the mechanical behavior and macrophage-mediated degradation of a novel, degradable, electrospun poly-urethane vascular graft.

J Mech Behav Biomed Mater 2020 12 3;112:104077. Epub 2020 Sep 3.

Center for Biomedical Research, Medical University of Vienna, Vienna, Austria; Ludwig Boltzmann Institute for Cardiovascular Research, Vienna, Austria. Electronic address:

An assessment tool to evaluate the degradation of biodegradable materials in a more physiological environment is still needed. Macrophages are critical players in host response, remodeling and degradation. In this study, a cell culture model using monocyte-derived primary macrophages was established to study the degradation, macro-/micro-mechanical behavior and inflammatory behavior of a new designed, biodegradable thermoplastic polyurethane (TPU) scaffold, over an extended period of time in vitro. For in vivo study, the scaffolds were implanted subcutaneously in a rat model for up to 36 weeks. TPU scaffolds were fabricated via the electrospinning method. This technique provided a fibrous scaffold with an average fiber diameter of 1.39 ± 0.76 μm and an average pore size of 7.5 ± 1.1 μm. The results showed that TPU scaffolds supported the attachment and migration of macrophages throughout the three-dimensional matrix. Scaffold degradation could be detected in localized areas, emphasizing the role of adherent macrophages in scaffold degradation. Weight loss, molecular weight and biomechanical strength reduction were evident in the presence of the primary macrophage cells. TPU favored the switch from initial pro-inflammatory response of macrophages to an anti-inflammatory response over time both in vitro and in vivo. Expression of MMP-2 and MMP-9 (the key enzymes in tissue remodeling based on ECM modifications) was also evident in vitro and in vivo. This study showed that the primary monocyte-derived cell culture model represents a promising tool to characterize the degradation, mechanical behavior as well as biocompatibility of the scaffolds during an extended period of observation.
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http://dx.doi.org/10.1016/j.jmbbm.2020.104077DOI Listing
December 2020

Riboflavin-mediated photooxidation to improve the characteristics of decellularized human arterial small diameter vascular grafts.

Acta Biomater 2020 10 29;116:246-258. Epub 2020 Aug 29.

Medical University of Vienna, Center for Biomedical Research, Austria; Ludwig Boltzmann Institute for Cardiovascular Research, Vienna, Austria; Austrian Cluster for Tissue Regeneration, Vienna, Austria. Electronic address:

Vascular grafts with a diameter of less than 6 mm are made from a variety of materials and techniques to provide alternatives to autologous vascular grafts. Decellularized materials have been proposed as a possible approach to create extracellular matrix (ECM) vascular prostheses as they are naturally derived and inherently support various cell functions. However, these desirable graft characteristics may be limited by alterations of the ECM during the decellularization process leading to decreased biomechanical properties and hemocompatibility. In this study, arteries from the human placenta chorion were decellularized using two distinct detergents (Triton X-100 or SDS), which differently affect ECM ultrastructure. To overcome biomechanical strength loss and collagen fiber exposure after decellularization, riboflavin-mediated UV (RUV) crosslinking was used to uniformly crosslink the collagenous ECM of the grafts. Graft characteristics and biocompatibility with and without RUV crosslinking were studied in vitro and in vivo. RUV-crosslinked ECM grafts showed significantly improved mechanical strength and smoothening of the luminal graft surfaces. Cell seeding using human endothelial cells revealed no cytotoxic effects of the RUV treatment. Short-term aortic implants in rats showed cell migration and differentiation of host cells. Functional graft remodeling was evident in all grafts. Thus, RUV crosslinking is a preferable tool to improve graft characteristics of decellularized matrix conduits.
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http://dx.doi.org/10.1016/j.actbio.2020.08.037DOI Listing
October 2020

Expression of enzymes involved in polyunsaturated fatty acid synthesis in the stallion testis and epididymis.

Reprod Fertil Dev 2020 Jun;32(9):851-861

Artificial Insemination and Embryo Transfer, Department for Small Animals and Horses, Vetmeduni Vienna, Veterinärplatz 1, 1210 Vienna, Austria; and Corresponding author. Email:

The aim of the present study was to characterise key enzymes involved in polyunsaturated fatty acid (PUFA) synthesis in the testis and epididymis collected from 2-year-old healthy warmblood stallions (n=10). The mRNA expression of fatty acid synthase, the Δ9-, Δ6-, Δ5- and Δ4-desaturases and elongases 6, 5 and 2 (encoded by the fatty acid synthase (FASN), the stearoyl-CoA desaturase (SCD), the fatty acid desaturase 2 (FADS2), the fatty acid desaturase 1 (FADS1), the delta 4-desaturase, sphingolipid 1 (DEGS1), ELOVL fatty acid elongase 6(ELOVL6), ELOVL fatty acid elongase 5 (ELOVL5), ELOVL fatty acid elongase 2 (ELOVL2) genes respectively) was determined in equine testis and epididymis. All enzymes were present in testicular tissue and along the epididymis, but mRNA expression differed among localisations. The protein localisation of FADS1, FADS2 and ELOVL5 was determined by immunohistochemistry. In the testes, FADS1 was expressed in the germinal cells and ELOVL5 was expressed in germinal and Leydig cells; FADS2 was not detected. In the epididymis, FADS1 and FADS2 were expressed in the principal and basal cells, whereas ELOVL5 was found only in the principal cells of the caput. All three enzymes were present in epididymal vesicles secreted by an apocrine mechanism. These results suggest active PUFA metabolism during spermatogenesis and epididymal sperm maturation in stallions.
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http://dx.doi.org/10.1071/RD19342DOI Listing
June 2020

S100A4 mRNA-protein relationship uncovered by measurement noise reduction.

J Mol Med (Berl) 2020 05 15;98(5):735-749. Epub 2020 Apr 15.

Genomics Core Facility, VetCore, University of Veterinary Medicine, Veterinärplatz 1, A-1210, Vienna, Austria.

Intrinsic biological fluctuation and/or measurement error can obscure the association of gene expression patterns between RNA and protein levels. Appropriate normalization of reverse-transcription quantitative PCR (RT-qPCR) data can reduce technical noise in transcript measurement, thus uncovering such relationships. The accuracy of gene expression measurement is often challenged in the context of cancer due to the genetic instability and "splicing weakness" involved. Here, we sequenced the poly(A) cancer transcriptome of canine osteosarcoma using mRNA-Seq. Expressed sequences were resolved at the level of two consecutive exons to enable the design of exon-border spanning RT-qPCR assays and ranked for stability based on the coefficient of variation (CV). Using the same template type for RT-qPCR validation, i.e. poly(A) RNA, avoided skewing of stability assessment by circular RNAs (circRNAs) and/or rRNA deregulation. The strength of the relationship between mRNA expression of the tumour marker S100A4 and its proportion score of quantitative immunohistochemistry (qIHC) was introduced as an experimental readout to fine-tune the normalization choice. Together with the essential logit transformation of qIHC scores, this approach reduced the noise of measurement as demonstrated by uncovering a highly significant, strong association between mRNA and protein expressions of S100A4 (Spearman's coefficient ρ = 0.72 (p = 0.006)). KEY MESSAGES: • RNA-seq identifies stable pairs of consecutive exons in a heterogeneous tumour. • Poly(A) RNA templates for RT-qPCR avoid bias from circRNA and rRNA deregulation. • HNRNPL is stably expressed across various cancer tissues and osteosarcoma. • Logit transformed qIHC score better associates with mRNA amount. • Quantification of minor S100A4 mRNA species requires poly(A) RNA templates and dPCR.
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http://dx.doi.org/10.1007/s00109-020-01898-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7241963PMC
May 2020

Generation of Differentiating and Long-Living Intestinal Organoids Reflecting the Cellular Diversity of Canine Intestine.

Cells 2020 03 28;9(4). Epub 2020 Mar 28.

Division of Small Animal Internal Medicine, Department for Small Animals and Horses, University of Veterinary Medicine, 1210 Vienna, Austria.

Functional intestinal disorders constitute major, potentially lethal health problems in humans. Consequently, research focuses on elucidating the underlying pathobiological mechanisms and establishing therapeutic strategies. In this context, intestinal organoids have emerged as a potent in vitro model as they faithfully recapitulate the structure and function of the intestinal segment they represent. Interestingly, human-like intestinal diseases also affect dogs, making canine intestinal organoids a promising tool for canine and comparative research. Therefore, we generated organoids from canine duodenum, jejunum and colon, and focused on simultaneous long-term expansion and cell differentiation to maximize applicability. Following their establishment, canine intestinal organoids were grown under various culture conditions and then analyzed with respect to cell viability/apoptosis and multi-lineage differentiation by transcription profiling, proliferation assay, cell staining, and transmission electron microscopy. Standard expansion medium supported long-term expansion of organoids irrespective of their origin, but inhibited cell differentiation. Conversely, transfer of organoids to differentiation medium promoted goblet cell and enteroendocrine cell development, but simultaneously induced apoptosis. Unimpeded stem cell renewal and concurrent differentiation was achieved by culturing organoids in the presence of tyrosine kinase ligands. Our findings unambiguously highlight the characteristic cellular diversity of canine duodenum, jejunum and colon as fundamental prerequisite for accurate in vitro modelling.
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http://dx.doi.org/10.3390/cells9040822DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226743PMC
March 2020

Long Term Evaluation of Nanofibrous, Bioabsorbable Polycarbonate Urethane Grafts for Small Diameter Vessel Replacement in Rodents.

Eur J Vasc Endovasc Surg 2020 04 23;59(4):643-652. Epub 2019 Dec 23.

Centre for Biomedical Research, Medical University of Vienna, Vienna, Austria; Ludwig Boltzmann Cluster for Cardiovascular Research, Medical University of Vienna, Vienna, Austria. Electronic address:

Objective: Biodegradable materials for in situ vascular tissue engineering could meet the increasing clinical demand for sufficient synthetic small diameter vascular substitutes in aortocoronary bypass and peripheral vascular surgery. The aim of this study was to design a new degradable thermoplastic polycarbonate urethane (dPCU) with improved biocompatibility and optimal biomechanical properties. Electrospun conduits made from dPCU were evaluated in short and long term follow up and compared with expanded polytetrafluoroethylene (ePTFE) controls.

Methods: Both conduits were investigated prior to implantation to assess their biocompatibility and inflammatory potential via real time polymerase chain reaction using a macrophage culture. dPCU grafts (n = 28) and ePTFE controls (n = 28) were then implanted into the infrarenal abdominal aorta of Sprague-Dawley rats. After seven days, one, six, and 12 months, grafts were analysed by histology and immunohistochemistry (IHC) and assessed biomechanically.

Results: Anti-inflammatory signalling was upregulated in dPCU conduits and increased significantly over time in vitro. dPCU and ePTFE grafts offered excellent long and short term patency rates (92.9% in both groups at 12 months) in the rat model without dilatation or aneurysm formation. In comparison to ePTFE, dPCU grafts showed transmural ingrowth of vascular specific cells resulting in a structured neovessel formation around the graft. The graft material was slowly reduced, while the compliance of the neovessel increased over time.

Conclusion: The newly designed dPCU grafts have the potential to be safely applied for in situ vascular tissue engineering applications. The degradable substitutes showed good in vivo performance and revealed desirable characteristics such as biomechanical stability, non-thrombogenicity, and minimal inflammatory response after long term implantation.
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http://dx.doi.org/10.1016/j.ejvs.2019.11.004DOI Listing
April 2020

Ki-67/CD3 ratio in the diagnosis of chronic inflammatory enteropathy in dogs.

J Vet Intern Med 2020 Jan 11;34(1):92-97. Epub 2019 Dec 11.

Department for Companion Animals and Horses, Clinic for Small Animal Internal Medicine, University of Veterinary Medicine, Vienna, Austria.

Background: T cells play a key role in the pathogenesis of chronic inflammatory enteropathy (CIE) in dogs. Cluster of differentiation 3 (CD3) antigen serves as a marker for T cells. In human medicine, Ki-67 is an indicator for cell growth but there are only a few studies in dogs with CIE.

Objective: To investigate Ki-67 in relation to T cells as a marker for CIE in dogs.

Animals: Eleven dogs with CIE and 6 healthy beagle controls (CO).

Methods: Retrospective case-control study. Dogs were clinically assessed by the Canine Chronic Enteropathy Clinical Activity Index (CCECAI). Duodenal mucosal biopsy samples were endoscopically obtained for histopathologic examination by means of the World Small Animal Veterinary Association score. Double-labeled immunofluorescence was used to investigate colocalization of Ki-67 and CD3 in epithelium and lamina propria (LP) of villi and crypts.

Results: Dogs with CIE had significantly higher clinical score (median, 5.0; interquartile range [IQR], 3-7) compared to CO (all 0; P < .001). The Ki-67/CD3 double-positive cells were significantly increased in the LP of the crypt region of CIE dogs (0.63 cells/mm ; IQR, 0-0.54) versus CO (0.08 cells/mm ; IQR, 0-0.26; P = .044). A significant correlation was found between CCECAI and the Ki-67/CD3 ratio in the LP of the crypt region (r = 0.670; P = .012) in dogs with CIE.

Conclusions And Clinical Importance: The Ki-67/CD3 ratio is upregulated in the LP crypt region of dogs with CIE and it correlates with clinical severity. Therefore, Ki-67/CD3 could be a useful tool for detection of CIE.
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http://dx.doi.org/10.1111/jvim.15680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6979107PMC
January 2020

Early luteal phase progestin concentration influences endometrial function in pregnant mares.

Theriogenology 2019 Feb 19;125:236-241. Epub 2018 Nov 19.

Platform Artificial Insemination and Embryo Transfer, Department for Small Animals and Horses, Vetmeduni Vienna, Veterinärplatz 1, 1210, Vienna, Austria. Electronic address:

In the horse, it is still unclear if and to what extent low progestin concentration contributes to early conceptus loss. In the present study, we have investigated if reduced or elevated progestin concentration in the early luteal phase influences endometrial function and conceptus development. We hypothesized that reduced progestin concentration via delayed downregulation of endometrial progesterone receptors (PR) influences endometrial function in healthy fertile mares while progestin substitution does not. Genitally healthy estrous mares (n = 8; age 4-14 years) were inseminated and treated with either altrenogest (0.044 mg/kg once daily orally) on days 5-10 after ovulation (ALT), cloprostenol (125 μg once daily intramuscularly) on days 0-3 after ovulation (CLO) or left untreated (CON). ALT and CLO treatment were chosen to increase and decrease total peripheral progestin concentration, respectively. Each treatment was given to every mare in consecutive cycles. On day 14 after ovulation, endometrial fluid was collected with a cotton roll inserted into the uterus and an endometrial biopsy for immunohistological demonstration of progesterone (PR) receptor distribution was collected. In endometrial fluid, free amino acid concentrations were analyzed by ion exchange liquid chromatography with an amino acid analyzer. Cell nuclei staining positive for the PR were determined in the luminal and glandular epithelium as well as in the stroma. Pregnancy rate tended to differ among treatments. The percentage of luminal epithelial cells staining positive for PR differed among treatments (p < 0.05) and was higher in CLO (84.1 ± 1.9%) than in ALT (70.7 ± 4.7%) and CON cycles (72.8 ± 4.1%). Concentrations of the amino acids isoleucine (CON 0.17 ± 0.03, CLO 0.14 ± 0.02, ALT 0.23 ± 0.04 μmol) and lysine (CON 0.27 ± 0.08, CLO 0.18 ± 0.05, ALT 0.44 ± 0.13 μmol) were influenced by treatment (p < 0.05) and lower in CLO than in ALT and CON cycles. In conclusion, impaired luteal function due to CLO treatment during the early luteal phase of pregnant mares delayed downregulation of progesterone receptors in the endometrial epithelium on day 14. This influenced endometrial function as reflected in lower concentrations of the amino acids lysine and isoleucine in endometrial secretions. Enhanced progestin concentration had less clear effects in healthy fertile mares.
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http://dx.doi.org/10.1016/j.theriogenology.2018.11.018DOI Listing
February 2019

Intussusceptive Pillar Formation in Developing Porcine Glomeruli.

J Vasc Res 2018 13;55(5):278-286. Epub 2018 Sep 13.

Department of Morphology, Faculty of Veterinary Medicine, Ghent University, Merelbeke,

Background/aims: Intussusceptive angiogenesis (IA) is a dynamic process which contributes to vascular expansion and remodeling. Intraluminal pillars have long been the distinctive structural indicator of IA. However, the mechanism of their formation has not been fully elucidated.

Methods: Using light and electron microscopy, we studied intussusceptive vascular growth in the developing porcine metanephric kidney.

Results: We observed intraluminal pillars formed by endothelial cells in the vasculature of developing glomeruli. Their diameter was < 2.5 µm, consistent with the diameter of nascent pillars. TEM revealed that the majority of these pillars consisted only of endothelium. However, a central core of extracellular matrix (ECM) covered by endothelium, reminiscent of a more mature intussusceptive pillar, was also found in the lumen of a glomerular capillary. Perivascular cells or pericytes were not involved in the pillar structure during these stages of formation.

Conclusion: This study shows ECM presence in a mature intussusceptive pillar without any perivascular cell involvement in the structure. This leads to the hypothesis that ECM deposition precedes the participation of these cells in the formation of intraluminal pillars during IA in porcine metanephric glomerular capillaries.
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http://dx.doi.org/10.1159/000490905DOI Listing
September 2019

Influences of intrauterine semen administration on regulatory T lymphocytes in the oestrous mare (Equus caballus).

Theriogenology 2018 Sep 1;118:119-125. Epub 2018 Jun 1.

Platform Artificial Insemination and Embryo Transfer, Department for Small Animals and Horses, Vetmeduni Vienna, Veterinärplatz 1, 1210, Vienna, Austria. Electronic address:

In the mare, early pregnancy loss is common, but involvement of the maternal immune system in the pathogenesis of this condition has not been investigated in detail so far. In the present study, we assessed effects of exposure of the endometrium to semen or seminal plasma in oestrous mares on the response of regulatory T lymphocytes (Tregs) in the peripheral circulation as well as in the endometrium. Raw semen, seminal plasma or PBS (control) were introduced into the uterus of oestrous mares (n = 12). Blood was collected immediately before insemination or PBS infusion (time 0), and 12, 24 and 48 h thereafter. Endometrial biopsies were collected at 24 h. In peripheral blood, Treg (CD4Foxp3) cells were determined by flow cytometry. In endometrial biopsies, Tregs were assessed as cells staining positive for Foxp3 by immunohistochemistry. The percentage of Tregs in blood decreased (p < 0.05) at 12 h after exposure to seminal plasma, tended to decrease in response to raw semen (p = 0.095) but not to PBS. Leukocyte and PMN counts were not affected. In the endometrium, numbers of Foxp3 positive cells at 24 h after insemination or PBS infusion were not changed by treatment. Results of the present study provide only little evidence that maternal tolerance of pregnancy in the horse is modulated already by exposure of the oestrous endometrium to seminal plasma at mating.
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http://dx.doi.org/10.1016/j.theriogenology.2018.05.030DOI Listing
September 2018

Acellular vascular matrix grafts from human placenta chorion: Impact of ECM preservation on graft characteristics, protein composition and in vivo performance.

Biomaterials 2018 09 29;177:14-26. Epub 2018 May 29.

Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna, Austria; Center for Biomedical Research Medical University of Vienna, Vienna, Austria; Austrian Cluster for Tissue Regeneration, Vienna, Austria. Electronic address:

Small diameter vascular grafts from human placenta, decellularized with either Triton X-100 (Triton) or SDS and crosslinked with heparin were constructed and characterized. Graft biochemical properties, residual DNA, and protein composition were evaluated to compare the effect of the two detergents on graft matrix composition and structural alterations. Biocompatibility was tested in vitro by culturing the grafts with primary human macrophages and in vivo by subcutaneous implantation of graft conduits (n = 7 per group) into the flanks of nude rats. Subsequently, graft performance was evaluated using an aortic implantation model in Sprague Dawley rats (one month, n = 14). In situ graft imaging was performed using MRI angiography. Retrieved specimens were analyzed by electromyography, scanning electron microscopy, histology and immunohistochemistry to evaluate cell migration and the degree of functional tissue remodeling. Both decellularization methods resulted in grafts of excellent biocompatibility in vitro and in vivo, with low immunogenic potential. Proteomic data revealed removal of cytoplasmic proteins with relative enrichment of ECM proteins in decelluarized specimens of both groups. Noteworthy, LC-Mass Spectrometry analysis revealed that 16 proteins were exclusively preserved in Triton decellularized specimens in comparison to SDS-treated specimens. Aortic grafts showed high patency rates, no signs of thrombus formation, aneurysms or rupture. Conduits of both groups revealed tissue-specific cell migration indicative of functional remodeling. This study strongly suggests that decellularized allogenic grafts from the human placenta have the potential to be used as vascular replacement materials. Both detergents produced grafts with low residual immunogenicity and appropriate mechanical properties. Observed differences in graft characteristics due to preservation method had no impact on successful in vivo performance in the rodent model.
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http://dx.doi.org/10.1016/j.biomaterials.2018.05.045DOI Listing
September 2018

Comparative proteome analysis of monolayer and spheroid culture of canine osteosarcoma cells.

J Proteomics 2018 04 11;177:124-136. Epub 2018 Jan 11.

Institute of Anatomy, Histology and Embryology, University of Veterinary Medicine Vienna, Veterinärplatz 1, A-1210 Vienna, Austria; VetCore Facility for Research, University of Veterinary Medicine Vienna, Veterinärplatz 1, A-1210 Vienna, Austria. Electronic address:

Osteosarcoma is an aggressive bone tumor with high metastasis rate in the lungs and affects both humans and dogs in a similar way. Three-dimensional tumor cell cultures mimic the in vivo situation of micro-tumors and metastases and are therefore better experimental in vitro models than the often applied two-dimensional monolayer cultures. The aim of the present study was to perform comparative proteomics of standard monolayer cultures of canine osteosarcoma cells (D17) and three-dimensional spheroid cultures, to better characterize the 3D model before starting with experiments like migration assays. Using DIGE in combination with MALDI-TOF/TOF we found 27 unique canine proteins differently represented between these two culture systems, most of them being part of a functional network including mainly chaperones, structural proteins, stress-related proteins, proteins of the glycolysis/gluconeogenesis pathway and oxidoreductases. In monolayer cells, a noticeable shift to more acidic pI values was noticed for several proteins of medium to high abundance; two proteins (protein disulfide isomerase A3, stress-induced-phosphoprotein 1) showed an increase of phosphorylated protein species. Protein distribution within the cells, as detected by immunohistochemistry, displayed a switch of stress-induced-phosphoprotein 1 from the cytoplasm (in monolayer cultures) to the nucleus (in spheroid cultures). Additionally, Western blot testing revealed upregulated concentrations of metastasin (S100A4), triosephosphate isomerase 1 and septin 2 in spheroid cultures, in contrast to decreased concentrations of CCT2, a subunit of the T-complex. Results indicate regulation of stress proteins in the process of three-dimensional organization characterized by a hypoxic and nutrient-deficient environment comparable to tumor micro-metastases.

Significance: Osteosarcoma is an aggressive bone tumor that early spreads to the lungs. Three-dimensional tumor cell cultures represent the avascular stage of micro-tumors and metastases, and should therefore represent a better experimental in vitro model compared to two-dimensional monolayer cultures. Significant differences have been reported in response to drug and radiation treatment between these two culture systems. A gel-based proteomic investigation was performed to compare protein patterns of a canine osteosarcoma cell line cultivated under those two conditions, to learn more about altered cell composition and its impact on cell behaviour. Due to the fact that the canine osteosarcoma is an accepted model for the human disease, results will be relevant for the human species as well.
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http://dx.doi.org/10.1016/j.jprot.2018.01.006DOI Listing
April 2018

Bioabsorption and Bioaccumulation of Cadmium in the Straw and Grain of Maize (Zea mays L.) in Growing Soils Contaminated with Cadmium in Different Environment.

Int J Environ Res Public Health 2017 11 16;14(11). Epub 2017 Nov 16.

Instituto de Investigaciones Agropecuarias INIA, Avenida Vicente Méndez 515, Chillán 3800062, Chile.

There is a worldwide increase of heavy metal or potentially toxic element (PTE), contamination in agricultural soils caused mainly by human and industrial action, which leads to food contamination in crops such as in maize. Cadmium (Cd) is a PTE often found in soils and it is ingested through food. It is necessary to determine the bioabsorption, distribution, and accumulation levels in maize to reduce or prevent food chain contamination. Cadmium absorption and accumulation in three maize cultivars were evaluated in three agricultural environments in Chile by increasing CdCl₂ rates (0, 1, and 2 mg·kg). Evaluation included Cd accumulation and distribution in different plant tissues, bioaccumulation factor (BAF), bioconcentration factor (BCF), translocation factor (TF), and tolerance index (TI). Cadmium whole-plant uptake was only affected by the CdCl₂ rate; the highest uptake was obtained with 2 mg·kg CdCl₂ (34.4 g·ha) ( < 0.05). Cadmium distribution in the maize plant usually exhibited the highest accumulation in the straw ( < 0.05), independently of the environment, Cd rate, and evaluated cultivar. Given the results for TF (TF > 2) and BAF (BAF > 1), the Los Tilos and Chillán environments were classified as having a high capacity to contaminate the food chain for all evaluated cultivars.
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http://dx.doi.org/10.3390/ijerph14111399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5708038PMC
November 2017

Ezrin and moesin expression in canine and feline osteosarcoma.

Histol Histopathol 2017 Aug 30;32(8):805-816. Epub 2016 Nov 30.

Institute of Anatomy, Histology and Embryology, University of Veterinary Medicine, Vienna, Austria.

Biological features of canine osteosarcomas (OS) differ markedly from those found in feline and resemble more human osteosarcomas, in particular for their high rate of metastasis and poor prognosis. Ezrin, radixin and moesin are members of the ERM protein family and link the actin cytoskeleton with the cell membrane. Ezrin and moesin have been shown to be of prognostic significance in tumor progression due to their role in the metastatic process. The objective of this study was to analyze ezrin and moesin protein expression in a series of dog (n = 16) and cat (n = 8) osteosarcoma samples using immunohistochemistry and western blot techniques. We found that cat OS have a higher moesin expression compared to dog OS, however, the active phosphorylated forms of moesin and ezrin Tyr353 were more abundant in the dog samples. A statistically significant difference was found for the low and high immunohistochemical scores of ezrin and pan-phospho-ERM proteins between cat and dog. Although phospho-ezrin Thr567 was higher in feline OS, the membranous localization in dog OS samples indicates the presence of the biologically active form. Therefore, the observed differences in phosphorylated forms of ezrin and moesin status should be further studied to demonstrate if they are relevant for different biological behavior between dog and cat OS.
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http://dx.doi.org/10.14670/HH-11-848DOI Listing
August 2017

Biocompatibility Assessment of a New Biodegradable Vascular Graft via In Vitro Co-culture Approaches and In Vivo Model.

Ann Biomed Eng 2016 Nov 7;44(11):3319-3334. Epub 2016 Apr 7.

Division of Biomedical Research, Medical University of Vienna, Vienna, Austria.

Following the implantation of biodegradable vascular grafts, macrophages and fibroblasts are the major two cell types recruited to the host-biomaterial interface. In-vitro biocompatibility assessment usually involves one cell type, predominantly macrophages. In this study, macrophage and fibroblast mono- and co-cultures, in paracrine and juxtacrine settings, were used to evaluate a new biodegradable thermoplastic polyurethane (TPU) vascular graft. Expanded-polytetrafluoroethylene (ePTFE) grafts served as controls. Pro/anti-inflammatory gene expression of macrophages and cytokines was assessed in vitro and compared to those of an in vivo rat model. Host cell infiltration and the type of proliferated cells was further studied in vivo. TPU grafts revealed superior support in cell attachment, infiltration and proliferation compared with ePTFE grafts. Expression of pro-inflammatory TNF-α/IL-1α cytokines was significantly higher in ePTFE, whereas the level of IL-10 was higher in TPU. Initial high expression of pro-inflammatory CCR7 macrophages was noted in TPU, however there was a clear transition from CCR7 to anti-inflammatory CD163 expression in vitro and in vivo only in TPU, confirming superior cell-biomaterial response. The co-culture models, especially the paracrine model, revealed higher fidelity to the immunomodulatory/biocompatibility behavior of degradable TPU grafts in vivo. This study established an exciting approach developing a co-culture model as a tool for biocompatibility evaluation of degradable biomaterials.
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http://dx.doi.org/10.1007/s10439-016-1601-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5093217PMC
November 2016

Effects of periovulatory gonadotrophin treatment on luteal function and endometrial expression of selected genes in cyclic pony mares.

Theriogenology 2016 Dec 15;86(9):2147-2155. Epub 2016 Jul 15.

Artificial Insemination and Embryo Transfer, Department for Small Animals and Horses, Vetmeduni Vienna, Vienna, Austria.

Progestin concentration in plasma during the early luteal phase is crucial for endometrial function and conceptus development. We hypothesized that periovulatory gonadotrophin treatment via support of luteal function affects endometrial gene expression in horses. Effect of age was analyzed as well. Shetland mares (n = 8, age 4-25 years) were assigned to the following treatments during five consecutive cycles in alternating order following a cross-over design: treatment hCG/-: preovulatory injection of hCG, but no gonadotrophin injection at detection of ovulation, treatment -/hCG: no preovulatory gonadodrophin injection, but injection of hCG at detection of ovulation, treatment eCG/-: preovulatory injection of eCG, but no gonadotrophin injection at detection of ovulation, treatment -/eCG: no preovulatory gonadotrophin injection, but injection of eCG at detection of ovulation, treatment control: no treatment. Concentration of progestin was analyzed by ELISA from the day of ovulation until Day 10. On Day 10, endometrial cells were collected transvaginally by cytobrush technique. Expression of mRNA of cyclooxygenase-2 (COX-2), prostaglandin F2α-synthase, prostaglandin E-synthase, progesterone receptor (PR), estradiol receptor (E2R), acyl-CoA-dehydrogenase (ACAD), uteroglobin (UGB), uteroferrin, and uterocalin was analyzed by RT qPCR. Immunohistological staining of endometrial tissue, obtained via biopsy, was performed for COX-2, PR and UGB. The P4 concentration was influenced by day of cycle (P < 0.01), but not by treatment. No effects of age on gene expression were determined. Neither of the periovulatory gonadotrophin treatments nor age influenced mRNA expression of the genes of interest. Treatment did also not affect immunohistological staining of the endometrium. In contrast, age affected the percentage of PR positive stromal cells (e.g. mare 1 (4 years): 65.5 ± 2.6, mare 2 (24 years): 82.7 ± 2.2%, P < 0.05) and COX-2 positive stained ciliated cells (e.g. mare 1: 15.8 ± 2.9, mare 2: 33.4 ± 6.0%, P < 0.05). In conclusion, no effects of periovulatory gonadotrophin treatment and age on endometrial gene expression in luteal phase pony mares were reported. A lack of treatment effects on luteal function and expression of PRs in the endometrium can at least in part be explained by differences in the reproductive physiology between horses and ponies.
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http://dx.doi.org/10.1016/j.theriogenology.2016.07.004DOI Listing
December 2016

Stem cell growth factor receptor in canine vs. feline osteosarcomas.

Oncol Lett 2016 Oct 12;12(4):2485-2492. Epub 2016 Aug 12.

Department of Pathobiology, University of Veterinary Medicine Vienna, A-1210 Vienna, Austria.

Osteosarcoma is considered the most common bone cancer in cats and dogs, with cats having a much better prognosis than dogs, since the great majority of dogs with osteosarcoma develop distant metastases. In search of a factor possibly contributing to this disparity, the stem cell growth factor receptor KIT was targeted, and the messenger (m)RNA and protein expression levels of KIT were compared in canine vs. feline osteosarcomas, as well as in normal bone. The mRNA expression of was quantified by reverse transcription-quantitative polymerase chain reaction, and was observed to be significantly higher in canine (n=14) than in feline (n=5) osteosarcoma samples (P<0.001). KIT protein expression was evaluated by immunohistochemistry, which revealed that 21% of canine osteosarcoma samples did not exhibit KIT staining in their neoplastic cells, while in 14% of samples, a score of 1 (<10% positive tumour cells) was observed, and in 50% and 14% of samples, a score of 2 (10-50% positivity) and 3 (>50% positivity), respectively, was observed. By contrast, the cancer cells of all the feline bone tumour samples analysed were entirely negative for KIT. Notably, canine and feline osteocytes of healthy bone tissue lacked any KIT expression. These results could be the first evidence that KIT may be involved in the higher aggressiveness of canine osteosarcoma compared with feline osteosarcoma.
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http://dx.doi.org/10.3892/ol.2016.5006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5038442PMC
October 2016

Anti-Muellerian hormone, inhibin A, gonadotropins, and gonadotropin receptors in bull calves after partial scrotal resection, orchidectomy, and Burdizzo castration.

Theriogenology 2017 Jan 7;87:242-249. Epub 2016 Sep 7.

Obstetrics, Gynecology and Andrology, Department for Small Animals and Horses, University of Veterinary Medicine, Vienna, Austria.

Eight-week-old calves were either castrated by partial scrotal resection (SR) without removing the testes (n = 10), Burdizzo (BZ) clamp (n = 10), orchidectomy (OR; n = 10), or were left gonad intact as controls (CO; n = 10). Concentrations of anti-Muellerian hormone (AMH), inhibin A, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) in plasma were determined from 16 to 48 weeks of age. At 18 months, testes of SR, BZ, and CO bulls were obtained and the immunolocalization of LH and FSH receptors and AMH analyzed. Concentration of AMH in plasma of CO and SR bulls decreased with increasing age (P < 0.001). A similar AMH profile in CO and SR indicates that SR did not induce a true cryptorchid state. In groups OR and BZ, AMH was undetectable. Plasma inhibin concentration was higher in groups CO and SR than BZ and OR (P < 0.001). Plasma LH and FSH concentrations decreased over time (P < 0.001) and were higher in groups BZ and OR than SR and CO (P < 0.001). In the testes, immunolabeling for AMH existed in Sertoli cells of CO and SR but not BZ bulls. FSH receptors were localized in Sertoli cells, Leydig cells, spermatocytes, and the epididymis of CO and SR animals, whereas LH receptors were restricted to Leydig cells. In BZ animals, FSH and LH receptors and AMH were absent, indicating complete testicular degeneration. In conclusion, AMH is a more reliable marker for the presence of testicular tissue in bulls than inhibin. Scrotal resection did not induce a true inguinal cryptorchid state but affected testicular responsiveness to gonadotropic stimulation.
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http://dx.doi.org/10.1016/j.theriogenology.2016.08.030DOI Listing
January 2017

Alternative lengthening of telomeres does exist in various canine sarcomas.

Mol Carcinog 2017 03 22;56(3):923-935. Epub 2016 Sep 22.

Department for Companion Animals and Horses, University of Veterinary Medicine Vienna, Vienna, Austria.

Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism (TMM) found in some human tumors such as sarcomas. Canine tumors are not characterized for ALT and the study aim was to identify if the ALT phenotype exists in canine sarcomas. Sixty-four canine sarcoma samples (20 snap-frozen, 44 FFPE) as well as six canine sarcoma cell lines were screened for ALT by C-circle assay. ALT was further evaluated by measuring telomere length via qPCR and telomere restriction-fragments including pulsed-field electrophoresis. ALT-associated proteins were validated by immunohistochemistry. Further, telomerase activity (TA) and gene expression were analyzed by TRAP and qPCR. DNA from 20 human neuroblastomas and 8 sarcoma cell lines served as comparative controls. ALT was detected in 9.4% (6/64) canine sarcomas including aggressive subtypes as hemangiosarcoma, osteosarcoma, and histiocytic sarcoma. C-circle levels were comparable with human ALT-positive controls. All ALT tumors demonstrated loss of ATRX expression and 5/6 showed strong p53 expression. TA was detected in 93% (14/15) snap-frozen samples including a sarcoma with ALT activity. This tumor showed long heterogeneous telomeres, and a high level of colocalization of DAXX with telomeres. One sarcoma was ALT and TA negative. All canine and human sarcoma cell lines were ALT negative. In this study, we demonstrated that canine sarcomas use ALT. As in humans, ALT was identified in aggressive sarcomas subtypes and coexisted with TA in one tumor. Overall, canine sarcomas seem to share many similarities with their human counterparts and appear an attractive model for comparative telomere research. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/mc.22546DOI Listing
March 2017

Establishment and Characterization of New Canine and Feline Osteosarcoma Primary Cell Lines.

Vet Sci 2016 Jun 1;3(2). Epub 2016 Jun 1.

Department of Pathobiology, Institute of Anatomy, Histology and Embryology, University of Veterinary Medicine, Veterinaerplatz 1, Vienna 1210, Austria.

Osteosarcomas are the most abundant form of bone malignancies in multiple species. Canine osteosarcomas are considered a valuable model for human osteosarcomas because of their similar features. Feline osteosarcomas, on the other hand, are rarely studied but have interesting characteristics, such as a better survival prognosis than dogs or humans, and less likelihood of metastasis. To enable experimental approaches to study these differences we have established five new canine osteosarcoma cell lines out of three tumors, COS_1186h, COS_1186w, COS_1189, and COS_1220, one osteosarcoma-derived lung metastasis, COS_1033, and two new feline osteosarcoma cell lines, FOS_1077 and FOS_1140. Their osteogenic and neoplastic origin, as well as their potential to produce calcified structures, was determined by the markers osteocalcin, osteonectin, tissue unspecific alkaline phosphatase, p53, cytokeratin, vimentin, and alizarin red. The newly developed cell lines retained most of their markers but only spontaneously formed spheroids produced by COS_1189 showed calcification .
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http://dx.doi.org/10.3390/vetsci3020009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5644629PMC
June 2016

Distribution and activity levels of matrix metalloproteinase 2 and 9 in canine and feline osteosarcoma.

Can J Vet Res 2016 Jan;80(1):66-73

Institute of Anatomy, Histology and Embryology (Gebhard, Walter), Institute of Pathology and Forensic Veterinary Medicine (Fuchs-Baumgartinger), VetCore Facility for Research (Razzazi-Fazeli), and Institute of Medical Biochemistry (Miller), University of Veterinary Medicine, Veterinaerplatz 1, A-1210 Vienna, Austria.

Overexpression of matrix metalloproteinases (MMPs) has been associated with increased tumor aggressiveness and metastasis dissemination. We investigated whether the contrasting metastatic behavior of feline and canine osteosarcoma is related to levels and activities of MMP2 and MMP9. Zymography and immunohistochemistry were used to determine expression levels of MMP2 and MMP9 in canine and feline osteosarcoma. Using immunohistochemistry, increased MMP9 levels were identified in most canine osteosarcomas, whereas cat samples more often displayed moderate levels. High levels of pro-MMP9, pro-MMP2, and active MMP2 were detected by gelatin zymography in both species, with significantly higher values for active MMP2 in canine osteosarcoma. These findings indicate that MMP2 is probably involved in canine and feline osteosarcoma and their expression and activity could be associated with the different metastatic behavior of canine and feline osteosarcoma.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4686036PMC
January 2016

Microsurgical and Percutaneous Epididymal Sperm Aspiration for Sperm Collection from Live Mice.

J Am Assoc Lab Anim Sci 2015 Sep;54(5):471-7

Institute of Laboratory Animal Science and Biomodels, University of Veterinary Medicine Vienna, Vienna, Austria.

Spermatozoa for in vitro fertilization of mouse oocytes and other methods of assisted reproduction typically are collected from the cauda epididymis of euthanized male mice. As an alternative to this terminal protocol, we developed and examined 2 methods for collecting sperm from anesthetized male mice without decreasing subsequent fertility: microsurgical epididymal sperm aspiration and, as a refinement, percutaneous epididymal sperm aspiration. Collected sperm was evaluated in terms of motility, concentration and in vitro fertilization ability. After recovery, both treated and untreated control male mice underwent in vivo fertility testing and subsequent histologic analysis of the treated male reproductive tract (epididymis and testis). In vitro fertilization using sperm recovered by the 2 collection methods was successfully achieved in all cases. The in vivo fertility test and the histologic analysis revealed no impairment of fertility and no permanent histologic alteration in the treated mice. Therefore, we recommend both techniques as simple and effective methods for recovering high-quality epididymal mouse sperm without having to euthanize fertile male mice.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4587614PMC
September 2015

Effect of stallion age on the expression of LH and FSH receptors and aromatase P450 in equine male reproductive tissues.

Reprod Fertil Dev 2016 Oct;28(12):2016-2026

Centre for Artificial Insemination and Embryo Transfer, University for Veterinary Sciences Vienna, Veterinaerplatz 1, 1210 Vienna, Austria.

The aim of the present study was to characterise receptors for LH and FSH (LHR and FSHR, respectively) and aromatase in epididymal and testicular tissue from stallions of different ages (prepubertal, young, mature and old). Gene and protein expression were assessed by real-time quantitative polymerase chain reaction (real-time qPCR), immunohistochemistry and multiple immunofluorescence labelling. There were no differences in LHR mRNA expression in epididymal and testicular parenchyma in stallions of different age. In contrast, expression of FSHR and CYP19A1 in caput, corpus and cauda epididymis and in testicular parenchyma increased with age (P<0.001). Immunolabelling for LHR, FSHR and aromatase was influenced by puberty. In postpubertal stallions, positive staining for LHR and aromatase was detected in Leydig cells, whereas protein expression of FSHR was present in Sertoli cells and primary spermatocytes. In prepubertal colts, staining for LHR, FSHR and aromatase was detected in seminiferous tubules. In epididymal tissue, aromatase was present in the cauda epididymis only, regardless of age. In conclusion, the results highlight the significance of gonadotropin action and oestrogen production for the maturation of male reproductive tissue in the horse. The presence of FSHR in the seminiferous tubules suggests effects of FSH on spermatogenesis in this species. The importance of oestrogen production for maintenance of testicular function in stallions was confirmed.
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http://dx.doi.org/10.1071/RD15027DOI Listing
October 2016
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