Characterisation of three novel canine osteosarcoma cell lines producing high levels of matrix metalloproteinases.

Three canine osteosarcoma cell lines were established from spontaneous pelvic and radial osteosarcomas. The cell populations cultured exhibited characteristics of malignancy and consisted of adherent, pleomorphic, mostly large spindle-shaped or polyhedral cells, characterised by the presence of numerous cytoplasmic granules and vacuoles. The main ultrastructural features included the presence of abundant rough endoplasmic reticulum and numerous cytoplasmic vesicles, deposit vacuoles and small cytoplasmic protrusions. Zymography showed that the cell lines produce high levels of MMP-2 and MMP-9, enzymes directly involved in crucial aspects of the metastatic process. Consistent with their osteoblastic lineage and malignant phenotype, all cell lines were immunoreactive to vimentin, osteopontin, PCNA, p53, MMP-2 and MMP-9, while they were negative for cytokeratin, desmin, SMA, Factor VIII, NSE, GFAP, Rb and p21 protein. No retroviral particles or RNA were detected ultrastructurally or with RT-PCR, although the possibility of viral involvement in osteosarcoma cannot be excluded. The new cell lines provide excellent in vitro models that may allow further studies on the pathobiology of canine osteosarcoma to be undertaken.

C2C12 co-culture on a fibroblast substratum enables sustained survival of contractile, highly differentiated myotubes with peripheral nuclei and adult fast myosin expression.

We describe a simple culture method for obtaining highly differentiated clonal C2C12 myotubes using a feeder layer of confluent fibroblasts, and document the expression of contractile protein expression and aspects of myofibre morphology using this system. Traditional culture methods using collagen- or laminin-coated tissue-culture plastic typically results in a cyclic pattern of detachment and reformation of myotubes, rarely producing myotubes of a mature adult phenotype. C2C12 co-culture on a fibroblast substratum facilitates the sustained culture of contractile myotubes, resulting in a mature sarcomeric register with evidence for peripherally migrating nuclei. Immunoblot analysis demonstrates that desmin, tropomyosin, sarcomeric actin, alpha-actinin-2 and slow myosin are detected throughout myogenic differentiation, whereas adult fast myosin heavy chain isoforms, members of the dystrophin-associated complex, and alpha-actinin-3 are not expressed at significant levels until >6 days of differentiation, coincident with the onset of contractile activity. Electrical stimulation of mature myotubes reveals typical and reproducible calcium transients, demonstrating functional maturation with respect to calcium handling proteins. Immunocytochemical staining demonstrates a well-defined sarcomeric register throughout the majority of myotubes (70-80%) and a striated staining pattern is observed for desmin, indicating alignment of the intermediate filament network with the sarcomeric register. We report that culture volume affects the fusion index and rate of sarcomeric development in developing myotubes and propose that a fibroblast feeder layer provides an elastic substratum to support contractile activity and likely secretes growth factors and extracellular matrix proteins that assist myotube development.

Renal pathology of polycystic kidney disease and concurrent hereditary nephritis in Bull Terriers.

OBJECTIVE: To describe the renal lesions in Bull Terrier polycystic kidney disease (BTPKD), to confirm that the renal cysts in BTPKD arise from the nephron or collecting tubule, and to identify lesions consistent with concurrent BTPKD and Bull Terrier hereditary nephritis (BTHN). DESIGN: Renal tissue from five Bull Terriers with BTPKD and eight control dogs was examined by light and transmission electron microscopy. Clinical data were collected from all dogs, and family history of BTPKD and BTHN for all Bull Terriers. RESULTS: In BTPKD the renal cysts were lined by epithelial cells of nephron or collecting duct origin that were usually squamous or cuboidal, with few organelles. They had normal junctional complexes, and basal laminae of varying thicknesses. Glomeruli with small, atrophic tufts and dilated Bowman's capsules, tubular loss and dilation, and interstitial inflammation and fibrosis were common. Whereas the lesions seen in BTHN by light microscope were nonspecific, the presence of characteristic ultrastructural glomerular basement membrane (GMB) lesions and a family history of this disease indicated concurrent BTHN was likely in three of five cases of BTPKD. CONCLUSION: This paper provides evidence that renal cysts in BTPKD are of nephron or collecting duct origin. In addition, GBM lesions are described that strongly suggest that BTPKD and BTHN may occur simultaneously.

Role of capsule in the pathogenesis of fowl cholera caused by Pasteurella multocida serogroup A.

We have constructed a defined acapsular mutant in Pasteurella multocida X-73 (serogroup A:1) by disrupting the hexA gene through the insertion of a tetracycline resistance cassette. The genotype of the hexA::tet(M) strain was confirmed by PCR and Southern hybridization, and the acapsular phenotype of this strain was confirmed by electron microscopy. The hexA::tet(M) strain was attenuated in both mice and chickens. Complementation of the mutant with an intact hexAB fragment restored lethality in mice but not in chickens. In contrast to the results described previously for P. multocida serogroup B (J. D. Boyce and B. Adler, Infect. Immun. 68:3463-3468, 2000), the hexA::tet(M) strain was sensitive to the bactericidal action of chicken serum, whereas the wild-type and complemented strains were both resistant. Following inoculation into chicken muscle, the bacterial count of the hexA::tet(M) strain decreased significantly, while the wild-type and complemented strains both grew rapidly over 4 h. The capsule is thus an essential virulence determinant in the pathogenesis of fowl cholera.

An apparent outbreak of cutaneous papillomatosis in merino sheep in patagonia, Argentina.

A retrospective study was performed on skin samples from an outbreak of cutaneous papillomatosis in Merino sheep that occurred in 1995. The samples were processed for routine histology, electron microscopy and immunocytochemistry for papilloma viruses. Particles of approximately 55 nm diameter were found in some nuclei of the stratum granulosum cells, while immunocytochemistry gave positive staining of cell nuclei in this layer. This study confirms that papillomas associated with papillomaviruses occur in sheep in Patagonia.

Comparison of the effects of Clostridium perfringens type D culture supernates in ligated intestinal loops of goats and sheep.

The effects of Clostridium perfringens type D culture supernates were compared in ligated loops of the small intestine (ileum) and colon of four goat kids and four lambs, the loops being examined histopathologically and electron microscopically 7 h after inoculation. No lesions were observed in the small intestine of any animal, or in control colonic loops. In the caprine and ovine colonic loops treated with culture supernates, most goblet cells were empty and the lumina contained a layer of mucus, polymorphonuclear leucocytes, bacteria and sloughed epithelial cells. The apical cytoplasm of the superficial epithelial cells was lost. Moderate oedema was observed in the submucosa and muscular layer. The colonic lesions were more severe in kids than in lambs. No changes were seen in vascular endothelial cells in any loop. 1999 W.B. Saunders and Company Ltd.