PDGFs and PDGFRs in canine osteosarcoma: New targets for innovative therapeutic strategies in comparative oncology

Platelet derived growth factor receptor (PDGFR)α and PDGFRβ are tyrosine kinase receptors that are overexpressed in 70–80% of human osteosarcomas (OSAs) and may be suitable therapeutic targets for specific kinase inhibitors (TKIs). Canine OSA shows histopathological and clinical features similar to human OSA, and is considered an excellent model in comparative oncology. This study investigated PDGF-A, PDGF-B, PDGFRα and PDGFRβ expression in 33 canine OSA samples by immunohistochemistry and in seven primary canine OSA cell lines by Western blot and quantitative PCR analysis.Immunohistochemical data showed that PDGF-A and PDGF-B are expressed in 42% and 60% of the OSAs analysed, respectively, while PDGFRα and PDGFRβ were expressed in 78% and 81% of cases, respectively. Quantitative PCR data showed that all canine OSA cell lines overexpressed PDGFRα, while 6/7 overexpressed PDGFRβ and PDGF-A relative to a normal osteoblastic cell line. Moreover, in vitro treatment with a specific PDGFR inhibitor, AG1296, caused a dose- and time-dependent decrease in AKT phosphorylation. Collectively, these data show that PDGFRs/PDGFs are co-expressed in canine osteosarcomas, which suggests that an autocrine and/or paracrine loop is involved and that they play an important role in the aetiology of OSA. PDGFRs may be suitable targets for the treatment of canine OSA with a specific TKI.     

Remote monitoring system as a tool for calving management in Mediterranean Buffalo heifers (Bubalus bubalis)

Buffalo breeding is common in Southern Italy. Dystocia compromises dam's and newborn health and welfare. Difficult parturition could be solved through prompt calving assistance, even if the identification of the beginning of delivery is challenging. Herein, we aimed to evaluate a remote calving alarm system in 15 Mediterranean buffalo heifers. An intravaginal probe was placed close to the external cervical os once premonitory signs of delivery were observed. No vaginal discharge nor signs of discomfort were notified in the days following the insertion of the probe. Heifers calved from 48 to 72 hr after the alarm was activated. The system correctly warned the farm personnel at the beginning of stage II of parturition, except for 2 cases. In the former, the intravaginal probe was expelled but the poor carrier network coverage negatively affected phone's signal quality; in the latter, recurrent vaginal prolapse was responsible for non-retention of the probe. Overall median expulsive phase was 68 ± 8 min, while the expulsion of a female calf took 54 ± 22.0 min and 90 ± 34.0 min in males, with significant difference (p =.02). Deliveries were homogeneously distributed across a 24-hr interval. No retention of foetal membranes nor metritis was identified at postpartum clinical examination. The calving alarm system used in this work was well tolerated in buffalo heifers. The introduction of smart technology in buffalo farming could contribute to the overall farm net return by reducing calf losses, especially for calves born from sexed-sorted semen, and by increasing animal welfare through quick resolution of dystocia. Further studies will be necessary to evaluate the net return in buffalo farms which will implement a remote calving alarm system on a wider population.     

In vitro and in vivo effects of toceranib phosphate on canine osteosarcoma cell lines and xenograft orthotopic models

Canine osteosarcoma (OSA) is the most common primary malignant bone tumour in dogs, and it has a high metastatic rate and poor prognosis. Toceranib phosphate (TOC; Palladia, Zoetis) is a veterinary tyrosine kinase inhibitor that selectively inhibits VEGFR‐2, PDGFRs and c‐Kit, but its efficacy is not yet fully understood in the treatment of canine OSA. Here, we evaluated the functional effects of TOC on six OSA cell lines by transwell, wound healing and colony formation assays. Subsequently, two cell lines (Wall and Penny) were selected and were inoculated in mice by intrafemoral injection to develop an orthotopic xenograft model of canine OSA. For each cell line, 30 mice were xenografted; half of them were used as controls, and the other half were treated with TOC at 40 mg/kg body weight for 20 days. TOC inhibited cell growth of all cell lines, but reduced invasion and migration was only observed in Penny and Wall cell lines. In mice engrafted with Penny cells and subjected to TOC treatment, decreased tumour growth was observed, and PDGFRs and c‐Kit mRNA were downregulated. Immunohistochemical analyses demonstrated a significant reduction of Ki67 staining in treated mice when compared to controls. The results obtained here demonstrate that TOC is able to slightly inhibit cell growth in vitro, while its effect is evident only in a Penny cell xenograft model, in which TOC significantly reduced tumour size and the Ki67 index without modifying apoptosis markers.     

Immune-metabolic-inflammatory markers in Holstein cows exposed to a nutritional and environmental stressing challenge

Dairy cows are exposed to multiple stressors during the productive cycle, such as metabolic challenges, overcrowding, grouping change, environmental stress and dietary errors. Thus, it is essential to study reliable markers able to detect stress conditions in dairy farms. This study evaluates dairy cows' immunologic and metabolic markers after the sudden and combined exposition to a high-grain diet (75% concentrates) and the abrupt change of the housing system (from free stall to tie stall). A group of twenty-four Holstein cows were enrolled in a challenge study of 28 days duration. Several immunological and metabolic blood markers were evaluated over the trial. Blood samples were taken at day 0 (normal value) and day 1, 3, 7, 14, 21, and 28 (challenge). Data were submitted to a mixed model for repeated measures, including time as fixed and cows as random effects. The nutritional and environmental challenge had heavy effects on animal welfare and cows responded with a dramatic rumination drop. Our results suggest that the most responsive markers after abiotic stressors in cows were as follows: Serum Amyloid A and ROM in the acute response; Ceruloplasmin and GGT in the mid acute and Albumin, Paroxonase and FRAP in the chronic phase. Serum Amyloid A, Ceruloplasmin, Paraoxonase, GGT and ROM resulted as positive phase proteins, while, Albumin and FRAP resulted as negative phase proteins. Preliminary obtained results could concur to develop strategies able to mitigate stressor effects; moreover, the proposed design can be used as a model to test stress nutritional modulators.     

A LAT mutation that inhibits T cell development yet induces lymphoproliferation

Mice homozygous for a single tyrosine mutation in LAT (linker for activation of T cells) exhibited an early block in T cell maturation but later developed a polyclonal lymphoproliferative disorder and signs of autoimmune disease. T cell antigen receptor (TCR)-induced activation of phospholipase C-gamma1 (PLC-gamma1) and of nuclear factor of activated T cells, calcium influx, interleukin-2 production, and cell death were reduced or abrogated in T cells from LAT mutant mice. In contrast, TCR-induced Erk activation was intact. These results identify a critical role for integrated PLC-gamma1 and Ras-Erk signaling through LAT in T cell development and homeostasis.     

Lack of a fusion requirement for development of bone marrow-derived epithelia

Analysis of developmental plasticity of bone marrow-derived cells (BMDCs) is complicated by the possibility of cell-cell fusion. Here we demonstrate that epithelial cells can develop from BMDCs without cell-cell fusion. We use the Cre/lox system together with beta-galactosidase and enhanced green fluorescent protein expression in transgenic mice to identify epithelial cells in the lung, liver, and skin that develop from BMDCs without cell fusion.     

Green tea modulates alpha(1)-adrenergic stimulated glucose transport in cultured rat cardiomyocytes

alpha1-Adrenergic stimulation triggers glucose transport in the heart through the translocation of glucose transporter (GLUT) 1 and GLUT4 to plasma membranes, mediated by protein kinase C (PKC) isoforms. Evidence is emerging that dietary polyphenolic compounds may act not only as antioxidants but also by modulating PKC-mediated signaling. This study evaluated the ability of a green tea extract (GTE) to modulate alpha1-adrenoceptor-mediated glucose transport in rat cardiomyocytes. GTE supplementation decreased phenylephrine (PhE)-stimulated glucose uptake and GLUT4 recruitment. PhE stimulation activated PKC alpha, beta, delta, and epsilon, while GTE supplementation decreased the translocation of beta and delta isoforms, but not alpha and epsilon, supporting the notion that GTE directly affects PKC activation and is a beta and delta isoform-selective PKC inhibitor. Due to reactive oxygen species (ROS) involvement in pathological heart alterations, the observation that GTE is able to both inhibit effects originated by some PKC isoforms and counteract ROS deleterious effects could be important in the prevention/counteraction of these diseases.     

Comparison of loss on ignition and thermal analysis stepwise methods for determination of sedimentary organic matter

• 1. Loss on ignition (LOI) is a widely used method to estimate organic matter (OM) in the sediment of marine and freshwater ecosystems. Thermogravimetric‐differential thermal analysis (TG‐DTA) of organic species provides information on thermal reactions, the amount and properties of clay structural water, organic species and carbonates.
• 2. The accuracy of LOI compared with that of TG‐DTA was evaluated in 33 sediment samples from the Lagoon of Venice (Italy).
• 3. In all tests conducted with TG‐DTA the mass loss of OM and the loss of clay structural water (LCSW) from oxidized samples were measured. The mass loss of OM at 350°C (TG‐DTA 350 OM) and the total extraction of organic matter at 567°C (TEOM) calculated from the difference between natural state samples and oxidized samples highlight the presence of both thermally labile and thermally stable substances.
• 4. The grain size data of sediment samples from the Lagoon shows a variable distribution between slightly muddy sand and mud. Loss of clay structural water at 350°C (LCSW 350) and total extraction of clay structural water at 567°C (TECSW) both estimated by TG‐DTA on oxidized samples, were found to correspond approximately to 6% and 10%, respectively of the clay fraction (<4 µm). This percentage may be used to correct LOI measurements of OM in sediments with high clay content.
• 5. LOI 350 (loss on ignition at 350°C) and LOI 550 (loss on ignition at 550°C) proved to be ～80% and ～200%, respectively, of total extraction of mass loss at 350°C (TG‐DTA 350 tot) and at 567°C (TEML) estimated by TG‐DTA on natural samples, meaning that the LOI 550 value represents a significant overestimate. The difference between the LOI 550 and TEML values indicates that the mass loss excess (MLE) may be accounted for by losses due to breakdown of carbonates.

Copyright © 2008 John Wiley & Sons, Ltd.