Hypoxia activates cadherin-22 synthesis via eIF4E2 to drive cancer cell migration, invasion and adhesion.

Hypoxia is a driver of cell movement in processes such as development and tumor progression. The cellular response to hypoxia involves a transcriptional program mediated by hypoxia-inducible factors, but translational control has emerged as a significant contributor. In this study, we demonstrate that a cell-cell adhesion molecule, cadherin-22, is upregulated in hypoxia via mTORC1-independent translational control by the initiation factor eIF4E2. We identify new functions of cadherin-22 as a hypoxia-specific cell-surface molecule involved in cancer cell migration, invasion and adhesion. Silencing eIF4E2 or cadherin-22 significantly impaired MDA-MB-231 breast carcinoma and U87MG glioblastoma cell migration and invasion only in hypoxia, while reintroduction of the respective exogenous gene restored the normal phenotype. Cadherin-22 was evenly distributed throughout spheroids and required for their formation and support of a hypoxic core. Conversely, E-cadherin translation was repressed by hypoxia and only expressed in the oxygenated cells of U87MG spheroids. Furthermore, immunofluorescence on paraffin-embedded human tissue from 40 glioma and 40 invasive ductal breast carcinoma patient specimens revealed that cadherin-22 expression colocalized with areas of hypoxia and significantly correlated with tumor grade and progression-free survival or stage and tumor size, respectively. This study broadens our understanding of tumor progression and metastasis by highlighting cadherin-22 as a potential new target of cancer therapy to disable hypoxic cancer cell motility and adhesion.

Release of endothelial cell associated VEGFR2 during TGF-ß modulated angiogenesis in vitro.

BACKGROUND: Sprouting angiogenesis requires vascular endothelial proliferation, migration and morphogenesis. The process is regulated by soluble factors, principally vascular endothelial growth factor (VEGF), and via bidirectional signaling through the Jagged/Notch system, leading to assignment of tip cell and stalk cell identity. The cytokine transforming growth factor beta (TGF-ß) can either stimulate or inhibit angiogenesis via its differential surface receptor signaling. Here we evaluate changes in expression of angiogenic signaling receptors when bovine aortic endothelial cells were exposed to TGF-ß1 under low serum conditions. RESULTS: TGF-ß1 induced a dose dependent inhibition of tip cell assignment and subsequent angiogenesis on Matrigel, maximal at 5.0 ng/ml. This occurred via ALK5-dependent pathways and was accompanied by significant upregulation of the TGF-ß co-receptor endoglin, and SMAD2 phosphorylation, but no alteration in Smad1/5 activation. TGF-ß1 also induced ALK5-dependent downregulation of Notch1 but not of its ligand delta-like ligand 4. Cell associated VEGFR2 (but not VEGFR1) was significantly downregulated and accompanied by reciprocal upregulation of VEGFR2 in conditioned medium. Quantitative polymerase chain reaction analysis revealed that this soluble VEGFR2 was not generated by a selective shift in mRNA isoform transcription. This VEGFR2 in conditioned medium was full-length protein and was associated with increased soluble HSP-90, consistent with a possible shedding of microvesicles/exosomes. CONCLUSIONS: Taken together, our results suggest that endothelial cells exposed to TGF-ß1 lose both tip and stalk cell identity, possibly mediated by loss of VEGFR2 signaling. The role of these events in physiological and pathological angiogenesis requires further investigation.

KRAS Mutations in Canine and Feline Pancreatic Acinar Cell Carcinoma.

Companion animals may serve as valuable models for studying human cancers. Although KRAS is the most commonly mutated gene in human ductal pancreatic cancers (57%), with mutations frequently occurring at codons 12, 13 and 61, human pancreatic acinar cell carcinomas (ACCs) lack activating KRAS mutations. In the present study, 32 pancreatic ACC samples obtained from 14 dogs and 18 cats, including seven metastases, were analyzed for six common activating KRAS mutations located in codons 12 (n = 5) and 13 (n = 1) using Sequenom MassARRAY. No KRAS mutations were found, suggesting that, similar to human pancreatic ACC, KRAS mutations do not play a critical role in feline or canine pancreatic ACC. Due to the similarity of the clinical disease in dogs and cats to that of man, this study confirms that companion animals offer potential as a suitable model for investigating this rare subtype of pancreatic carcinoma.

Receptor Tyrosine Kinase Expression Profiles in Canine Cutaneous and Subcutaneous Mast Cell Tumors.

The receptor tyrosine kinase (RTK) KIT is a major focus of current research into canine mast cell tumors (MCTs). Little is known about the role of other RTKs, such as vascular endothelial growth factor receptors (VEGFRs) and platelet-derived growth factor receptors (PDGFRs). These RTKs are dysregulated in many human and animal cancers and are key regulators of tumor angiogenesis. The aims of this study were to assess the expression and activation (phosphorylation) status of KIT, VEGFR2, and PDGFR (α and ß) in canine MCTs and to examine associations with various clinical outcomes. c-KITmutational status and KIT cellular localization pattern were also evaluated for these tumors. Twenty-seven MCTs, consisting of 5 subcutaneous and 22 cutaneous tumors, from 25 dogs were evaluated. MCT biopsies, cultured mast cells, and skin from the surgical margin were analyzed through Western blotting. MCT biopsies were also used for KIT immunohistochemical labeling and polymerase chain reaction for c-KITmutational analysis. MCT had heterogeneous expression profiles for all 3 RTKs, which varied in intensity and activation status. Statistical analyses showed phosphorylated KIT, VEGFR2, and KIT cellular localization to be predictive of decreased survival time, disease-free interval, and increased metastatic rate. Expression of VEGFR2 and KIT diffuse cytoplasmic labeling were also significantly associated with increased rate of local recurrence. The results of the study show that phosphorylated KIT, KIT, VEGFR2, and PDGFRß, in addition to KIT localization, may be valuable prognostic determinants in MCTs and should be further studied to improve diagnostic and therapeutic modalities.

Dichloroacetate affects proliferation but not survival of human colorectal cancer cells.

Dichloroacetate (DCA) is a metabolic reprogramming agent that reverses the Warburg effect, causing cancer cells to couple glycolysis to oxidative phosphorylation. This has been shown to induce apoptosis and reduce the growth of various types of cancer but not normal cells. Colorectal cancer cells HCT116, HCT116 p53(-/-), and HCT116 Bax(-/-), were treated with DCA in vitro. Response to treatment was determined by measuring PDH phosphorylation, apoptosis, proliferation, and cell cycle. Molecular changes associated with these responses were determined using western immunoblotting and quantitative PCR. Treatment with 20 mM DCA did not increase apoptosis, despite decreasing levels of anti-apoptotic protein Mcl-1 after 6 h, in any of the cell lines observed. Mcl-1 expression was stabilized with MG-132, an inhibitor of proteasomal degradation. A decrease in Mcl-1 correlated with a decrease in proliferation, both of which showed dose-dependence in DCA treated cells. Cells showed nuclear localization of Mcl-1, however cell cycle was unaffected by DCA treatment. These data suggest that a reduction in the prosurvival Bcl-2 family member Mcl-1 due to increased proteasomal degradation is correlated with the ability of DCA to reduce proliferation of HCT116 human colorectal cancer cells without causing apoptosis.

Canine subcutaneous mast cell tumors: cellular proliferation and KIT expression as prognostic indices.

Molecular assays are widely used to prognosticate canine cutaneous mast cell tumors (MCT). There is limited information about these prognostic assays used on MCT that arise in the subcutis. The aims of this study were to evaluate the utility of KIT immunohistochemical labeling pattern, c-KIT mutational status (presence of internal tandem duplications in exon 11), and proliferation markers--including mitotic index, Ki67, and argyrophilic nucleolar organizing regions (AgNOR)--as independent prognostic markers for local recurrence and/or metastasis in canine subcutaneous MCT. A case-control design was used to analyze 60 subcutaneous MCT from 60 dogs, consisting of 24 dogs with subsequent local recurrence and 12 dogs with metastasis, as compared to dogs matched by breed, age, and sex with subcutaneous MCT that did not experience these events. Mitotic index, Ki67, the combination of Ki67 and AgNOR, and KIT cellular localization pattern were significantly associated with local recurrence and metastasis, thereby demonstrating their prognostic value for subcutaneous MCT. No internal tandem duplication mutations were detected in exon 11 of c-KIT in any tumors. Because c-KIT mutations have been demonstrated in only 20 to 30% of cutaneous MCT and primarily in tumors of higher grade, the number of subcutaneous MCT analyzed in this study may be insufficient to draw conclusions on the role c-KIT mutations in these tumors.

Selenomethionine increases proliferation and reduces apoptosis in bovine mammary epithelial cells under oxidative stress.

The decline in mammary epithelial cell number as lactation progresses may be due, in part, to oxidative stress. Selenium is an integral component of several antioxidant enzymes. The present study was conducted to examine the effect of oxidative stress and selenomethionine (SeMet) on morphology, viability, apoptosis, and proliferation of bovine mammary epithelial cells (BMEC) in primary culture. Cells were isolated from mammary glands of lactating dairy cows and grown for 3 d in a low-serum gel system containing lactogenic hormones and 0 or 100 µM H2O2 with 0, 10, 20, or 50 nM SeMet. Hydrogen peroxide stress increased intracellular H2O2 to 3 times control concentrations and induced a loss of cuboidal morphology, cell-cell contact, and viability of BMEC by 25%. Apoptotic cell number more than doubled during oxidative stress, but proliferating cell number was not affected. Supplementation with SeMet increased glutathione peroxidase activity 2-fold and restored intracellular H2O2 to control levels with a concomitant return of morphology and viability to normal. Apoptotic BMEC number was decreased 76% below control levels by SeMet and proliferating cell number was increased 4.2-fold. These findings suggest that SeMet modulated apoptosis and proliferation independently of a selenoprotein-mediated reduction of H2O2. In conclusion, SeMet supplementation protects BMEC from H2O2-induced apoptosis and increased proliferation and cell viability under conditions of oxidative stress.

Canine subcutaneous mast cell tumor: characterization and prognostic indices.

Histologic grading schemes for canine cutaneous mast cell tumors (MCTs) were not developed for subcutaneous MCTs. Despite this, subcutaneous MCTs are currently categorized by many as grade II or higher. The aim of this investigation was to assess the pathology and clinical outcome for subcutaneous MCTs to provide a more accurate prognosis. Information on clinical outcome for 306 dogs was obtained from veterinarians and correlated with histologic features. Mean and median follow-up was 842 and 891 days, respectively (range, 3-2,305 days). Only 27 (9%) were confirmed as mast cell-related deaths. Metastasis occurred in 13 (4%), and 24 (8%) had local reoccurrence, even though 171 (56%) cases had incomplete surgical margins. Median survival time was not reached, and the estimated 6-month, 1-, 2-, and 5-year survival probabilities were 95%, 93%, 92%, and 86%, respectively. Dogs were euthanized or died as a result of local tumor reoccurrence, additional MCT development distant to the surgical site, or metastasis. Decreased survival time was linked to mitotic index (number of mitotic figures per 10 high-power fields), infiltrative growth pattern, and presence of multinucleation. Both univariable and multivariable analysis showed mitotic index to be strongly predictive of survival, local reoccurrence, and metastasis. The results of the study indicate that the majority of subcutaneous MCTs have a favorable prognosis, with extended survival times and low rates of reoccurrence and metastasis.

Selenomethionine stimulates expression of glutathione peroxidase 1 and 3 and growth of bovine mammary epithelial cells in primary culture.

This study examined the localization of cellular glutathione peroxidase (GPx1) and extracellular glutathione peroxidase (GPx3) in lactating mammary tissue and in primary cultures of bovine mammary epithelial cells (BMEC). The effect of selenium as selenomethionine (SeMet) on the growth and viability of BMEC and GPx protein expression and activity were also studied. Single mammary epithelial cells were recovered by serial collagenase/hyaluronidase digestion from lactating bovine mammary tissue and cultured in a low-serum collagen gel system enriched with lactogenic hormones and 0, 10, 20, or 50 nM SeMet. Positive immunostaining with anti-cytokeratin and bovine anti-casein confirmed the epithelial nature and differentiated state of BMEC. Addition of SeMet to media facilitated rapid confluence of BMEC and formation of dome structures. Immunohistochemical and immunocytochemical staining revealed that both GPx1 and GPx3 are synthesized by BMEC and localized in the cytoplasm and nucleus. Up to 50 nM SeMet linearly increased BMEC number and viability over 5 d of culture. Bovine mammary epithelial cells cultured in SeMet-supplemented medium also exhibited markedly elevated GPx activity and linear increases in abundance of GPx1 and GPx3 proteins. It is apparent that SeMet degradation to release Se for synthesis of selenoproteins is carried out by BMEC. Results indicate that bovine mammary epithelial cells express GPx1 and GPx3 in vivo and in vitro; SeMet enhances expression of these selenoproteins in vitro and the growth and viability of BMEC.

Low-dose metronomic cyclophosphamide treatment mediates ischemia-dependent K-ras mutation in colorectal carcinoma xenografts.

Antiangiogenic therapies are promising approaches to cancer control, but the details of their effects on subsequent tumor progression are not fully understood. Such therapies have the potential to eventually generate extensive amounts of tumor ischemia, and we previously demonstrated that ischemic conditions induce K-ras mutations in cells with deficient mismatch repair (MMR) mechanisms. This suggested that similar effects on oncogene mutagenesis may accompany antiangiogenic therapy. To test this, MMR-deficient colorectal cancer cells (Dks-8) were xenografted into immune-deficient mice and treated with the antiangiogenic regimen of low-dose/metronomic cyclophosphamide for 2 weeks followed by a 2-week recovery period without therapy. This treatment resulted in transient tumor growth inhibition, increased hypoxia, and decreased microvessel density, and cancer cells from treated tumors acquired activating mutations of the K-ras oncogene (K-ras(G13D)). In vitro exposure of Dks-8 cells to the active metabolite of cyclophosphamide (4-hydroxycyclophosphamide) had no effect on the K-ras status, indicating that there was no direct action of this alkylating agent on K-ras mutagenesis. In addition, cells sorted from hypoxic regions of Dks-8 tumors were enriched in K-ras(G13D) mutants. Collectively, our studies suggest that increases in tumor hypoxia induced by antiangiogenic treatment may lead to K-ras mutation and consequently tumor progression, especially in susceptible individuals.

Epithelial and fibroblast cell lines cultured from the transgenic BigBlue rat: an in vitro mutagenesis assay.

We have isolated, cultured, and immortalised three new BigBlue transgenic rat cell lines for the study of mutation induction in vitro. The two epithelial cell lines, from the mammary gland and oral cavity, were designated BBR/ME and BBR/OE, respectively, and the third is a mammary fibroblast line designated BBR/MFib. We have characterised these cell lines with respect to chromosome number and the expression of some cell-specific antigens. The clonogenic survival and cII transgene mutation induction responses of these three cell lines to N-ethyl-N-nitrosourea (ENU) treatment were determined. Both epithelial cell lines were much more sensitive to ENU toxicity than was the fibroblast cell line. However, all cell lines showed similar ENU dose-dependent increases in mutant frequency. We hope that cell lines such as these will extend the power of the BigBlue assay to in vitro studies.

Effects of relaxin on matrix remodeling enzyme activity of cultured equine ovarian stromal cells.

Relaxin participates in extracellular matrix (ECM) remodeling in many reproductive organs, including the ovary, by regulating proteolytic enzyme activity. Accumulated evidence indicates this action of relaxin is involved in ovarian follicle development and ovulation. Equine follicles are embedded in cortex that is at the center of the ovary and they must expand/emigrate to the fossa, the only site in the ovary for ovulation. Due to the tremendous expansion of the follicle in this species, we hypothesized that ovarian stromal remodeling would be extensive. Therefore, cultured equine ovarian stromal cell (EOSC) lines were obtained from stroma at the apex of large follicles and the effects of relaxin on gelatinases A and B, tissue inhibitors of matrix metalloproteinases (TIMPs), plasminogen activators (PAs) and PA inhibitor-1 (PAI-1) activities were assessed. Our results showed that equine relaxin increased the activity of total gelatinase A (both pro forms and mature forms) and latent progelatinase B present in conditioned medium, latent progelatinase A present in cell extracts, and TIMP-1 and TIMP-2 present in conditioned medium. This study also revealed that equine relaxin increased the urokinase-type PA activity in conditioned medium and cell extracts, tissue-type PA activity in ECM and PAI-1 activity in conditioned medium. These results suggest that relaxin may contribute to equine follicle growth and migration, and facilitate ovulation by modulating the degradation of ECM in ovarian stromal tissue.

Heterogeneous vascular dependence of tumor cell populations.

Cells within a tumor are highly heterogeneous with respect to a wide range of genotypic and phenotypic characteristics. The latter include such properties as growth, survival, invasion, and metastasis. We asked whether the degree to which individual tumor cells rely on a tumor's vasculature might also be heterogeneous. By adapting an intravital Hoechst 33342 staining technique, we labeled and isolated tumor cells based on their relative proximity to perfused vessels. Because tumor regions distal to the vasculature are likely hypoxic, we examined cells deficient for hypoxia-inducible factor-1alpha (HIF-1alpha), a transcription factor that has been shown to mediate hypoxia-induced responses, including apoptosis. Despite reduced vascularization in HIF-1alpha-/- embryonic stem cell-derived tumors, their growth in vivo was found to be accelerated relative to HIF-1alpha+/+ tumor counterparts. We hypothesized that this paradoxical observation is because of decreased apoptotic rate, resulting in diminished vascular dependence of HIF-1alpha-/- cells. Analysis of heterogeneous tumors established from mixtures of HIF-1alpha+/+ with HIF-1alpha-/- cells revealed that the proportion of cells expressing wild-type HIF-1alpha was increased in perivascular areas and decreased in distal tumor regions. Thus, cells expressing HIF-1alpha were found to be highly dependent on proximity to blood vessels for their growth and survival in vivo, whereas cells that had lost HIF-1alpha expression were much less so. Heterogeneity in angiogenesis dependence was also observed among cell subpopulations isolated from human melanoma xenografts. This potential for selection of less vascular-dependent tumor cell variants throughout the course of disease progression may have important implications for the long-term efficacy of anti-angiogenic therapy.

Neutrophil-platelet interactions and their relevance to bovine respiratory disease.

Respiratory disease is a serious and significant health problem for the bovine industry. Classically, the clinical and research focus has been on the putative causative agents and conditions, and their interactions with host inflammatory cells, particularly alveolar macrophages and blood neutrophils. There is, currently, growing acceptance of the concept that blood platelets play a primary role in the inflammatory process. This review explores the implications of such pro-inflammatory activity, especially in the context of neutrophil-platelet interactions, and the species specificity of cellular responses. The relevance of these issues for the treatment and prevention of bovine respiratory disease is also discussed.

Possible mechanisms of acquired resistance to anti-angiogenic drugs: implications for the use of combination therapy approaches.

The ultimate target of anti-angiogenic drugs is the genetically stable, activated endothelial cell of a newly forming tumor blood vessel, rather than the genetically unstable tumor cell population per se. This led to the notion that acquired resistance to such drugs may not develop as readily, if at all. While there is some evidence that this lack of resistance development may be the case for some direct-acting angiogenesis inhibitors, it is becoming apparent that resistance can develop over time to many types of angiogenesis inhibitors including, possibly, some direct inhibitors, especially when used as monotherapies. Possible mechanisms for such acquired or induced resistance include: (i) redundancy of pro-angiogenic growth factors when the drug used targets a single such growth factor or its cognate endothelial cell-associated receptor tyrosine kinase; (ii) the anti-apoptotic/pro-survival function of growth factors such as VEGF, which, in high local concentrations, can antagonize the pro-apoptotic effects of various angiogenesis inhibitors; (iii) epigenetic, transient upregulation, or induction, of various anti-apoptotic effector molecules in host-endothelial cells; and (iv) heterogeneous vascular dependence of tumor cell populations. It is suggested that long-term disease control with anti-angiogenic drugs can be best achieved by judicious combination therapy. In this regard, the great molecular diversity of anti-angiogenic drug targets, in contrast to chemotherapy, makes this a particularly attractive therapeutic option, especially when approved, commercially available drugs considered to have anti-angiogenic effects are used in such combination treatment strategies.

Effects of human IL-8 isoforms on bovine neutrophil function in vitro.

Interleukin 8 (IL-8) is a potent chemotactic and activating agent for human neutrophils and bovine IL-8 is chemotactic for bovine neutrophils; however, it is unclear whether IL-8 activates bovine neutrophils. Two isoforms of human recombinant (hr) IL-8 protein (77 and 72 amino acid) were used to stimulate bovine neutrophils in vitro. Bovine neutrophils exhibited significant migration in the presence of 0.1, 0.5, 1.0 and 5.0ngml(-1) hr IL-8 when incubated for 30min at 37 degrees C in a modified Boyden chamber assay. Both the 77 and 72 aa forms were equally effective in inducing migration in this assay. At the highest doses of IL-8 examined (1 and 5ngml(-1)), migration was similar to migration in the presence of 20% zymosan-activated serum (ZAS) or 12h lipopolysaccharide (LPS)-stimulated blood monocyte supernatants (CM). Significant (p<0. 05) release of alkaline phosphatase (ALK-P) (from specific granules) occurred but myeloperoxidase (MPO) release and superoxide anion production were not enhanced in bovine neutrophils by either form of hrIL-8 at any of the doses tested. Significant (p<0.05) alkaline phosphatase release was observed in the presence of 10 and 100ngml(-1) for the 72 aa form of IL-8 and only at the higher dose for the 77 aa form of IL-8. The ZAS and CM significantly enhanced neutrophil degranulation of ALK-P and MPO as well as inducing superoxide anion production. These results suggest that IL-8 may play a role in both neutrophil recruitment and activation during bovine inflammatory processes.

Production of gelatinases and tissue inhibitors of matrix metalloproteinases by equine ovarian stromal cells In vitro.

Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) play very important roles in extracellular matrix (ECM) remodeling in ovarian follicle growth and ovulation. Equine follicles are embedded in cortex that is at the center of the ovary, and they must expand/emigrate to the fossa, the only site in the ovary for ovulation. Therefore, equine ovarian stromal cells (EOSC) are probably involved in ECM remodeling during follicle growth. This study examined whether cultured EOSC synthesize gelatinases and TIMPs, molecules essential for ECM remodeling in other systems. Results showed that cultured EOSC (passage 3-8) had a fibroblast-like morphology and were positive for alpha-smooth muscle actin and type I procollagen by immunostaining. Gelatinase A (MMP-2), gelatinase B (MMP-9), TIMP-1, and TIMP-2 were present in EOSC-conditioned medium, and TIMP-3 in ECM of EOSC. Transforming growth factor beta significantly stimulated the activity of gelatinases A and B and TIMP-1 in conditioned medium from EOSC (p < 0.05). Phorbol 12-myristate 13-acetate also significantly stimulated the activity of gelatinases A and B and TIMP-1 in conditioned medium and of TIMP-3 in ECM (p < 0.05). Our results suggest that EOSC produce important components of the ECM remodeling machinery and, therefore, may play a role in the ECM remodeling during follicle growth in this species.

A preliminary study on the usefulness of huIL-8 in cervical relaxation of the ewe for artificial insemination and for embryo transfer.

The efficacy of using human interleukin 8 (huIL-8) as an agent for inducing cervical relaxation in estrous and diestrous sheep was assessed in a small pilot study. Multiparous, estrus-synchronized ewes were treated for either 2 or 5 consecutive days with vaginal suppositories with or without 5 micrograms cytokine. Cervical penetration with an insemination instrument was then assessed in vivo. After euthanasia, physical, histological and enzymological properties of the cervix were examined. Treatment of diestrous sheep with huIL-8 did not result in recruitment of neutrophils into the cervix. Treatment of estrous sheep with huIL-8 usually led to neutrophil recruitment to the cervix and to either full or partial penetration of the cervix. However, some animals receiving placebo treatment had neutrophil infiltration of both the vagina and cervix and, in one of these, partial penetration of the cervix was also achieved. Thus, treatment with IL-8 as the sole agent in the vaginal suppository was not sufficient to relax the cervix of the nonpregnant ewe in this study.

Blood vessel density in canine osteosarcoma.

Canine osteosarcoma is a prevalent bone neoplasm which has similarities to the human disease. We used a retrospective study to investigate the possibility that tumor vascularity may provide useful prognostic information, indicative of the role of this parameter in progression of this cancer. We quantified microvessel density in 52 histological specimens of primary tumor, immunostained for von Willebrand's Factor to identify vascular endothelium. For the 20 cases not euthanized at presentation or lost to follow-up, we found significantly higher tumor microvascular densities in animals presenting with detectable pulmonary metastases (5 of 20), and significantly lower densities in animals without metastatic disease at presentation, but later surviving to develop pulmonary metastases (7 of 20; P < 0.05). Animals with no evidence of pulmonary metastases at time of death (8 of 20) had intermediate vascular densities in their tumors. The results of this preliminary study suggest that vascularity of the primary tumor may be an indication of tumor progression. Future studies with a larger number of cases should establish whether vascular density can be a useful prognostic parameter for canine osteosarcoma.

Bovine platelet adhesion is enhanced by leukotoxin and sialoglycoprotease isolated from Pasteurella haemolytica A1 cultures.

Platelet and fibrin deposits are among characteristic changes observed in lung alveoli of cattle with pasteurellosis induced by Pasteurella haemolytica (biotype A, serotype 1). To determine whether the platelet function could be directly affected by protein products produced by the bacterium, the effects of leukotoxin and O-sialoglycoprotease, culture supernatant antigen secreted by Pasteurella haemolytica A1, on bovine platelet activation were examined by evaluating the enhancement of platelet adhesion to a negatively charged surface relative to untreated control samples. The glycoprotease, or the leukotoxin, was added to plasma free suspensions of bovine platelets and platelet adhesion assessed by two parameters: (i) the number of 3H-adenine-labeled adherent platelets and (ii) the morphology of unlabeled platelets adhering to the charged surface under scanning electron microscopy (SEM). In the presence of calcium, the glycoprotease produced a dose-dependent increase in adhesion. At a concentration of 4.0 micrograms glycoprotease extract protein per 10(7) platelets, a 2-fold increase in adhesion was observed which was similar to the increase in adhesion induced by 0.10 units of thrombin, a known platelet agonist. Both increased platelet adhesion and platelet aggregation were observed with 0.8 microgram glycoprotease extract protein in the presence of calcium. The response of the bovine platelet suspensions to leukotoxin extract protein was dependent on the dosage of the leukotoxin. Adhesion was enhanced at dosages of 25 micrograms leukotoxin protein per 10(7) platelets and below, while at dosages of 50 micrograms and above adhesion was suppressed. Thus, the two proteins secreted by P. haemolytica may interact directly with bovine platelets to initiate platelet aggregation and fibrin formation in alveolar tissue in pneumonic pasteurellosis.