Morphological and Immunohistochemical Characterization of Canine Osteosarcoma Spheroid Cell Cultures

Spheroid cell culture emerges as powerful in vitro tool for experimental tumour research. In this study, we established a scaffold‐free three‐dimensional spheroid system built from canine osteosarcoma (OS) cells (D17). Spheroids (7, 14 and 19 days of cultivation) and monolayer cultures (2 and 7 days of cultivation) were evaluated and compared on light and electron microscopy. Monolayer and spheroid cultures were tested for vimentin, cytokeratin, alkaline phosphatase, osteocalcin and collagen I by means of immunohistochemistry. The spheroid cell culture exhibited a distinct network of collagen I in particular after 19‐day cultivation, whereas in monolayer cultures, collagen I was arranged as a lamellar basal structure. Necrotic centres of large spheroids, as observed in 14‐ and 19‐day cultures, were characterized by significant amounts of osteocalcin. Proliferative activity as determined by Ki‐67 immunoreactivity showed an even distribution in two‐dimensional cultures. In spheroids, proliferation was predominating in the peripheral areas. Metastasis‐associated markers ezrin and S100A4 were shown to be continuously expressed in monolayer and spheroid cultures. We conclude that the scaffold‐free spheroid system from canine OS cells has the ability to mimic the architecture of the in vivo tumour, in particular cell–cell and cell–matrix interactions.     

Infection of a canine macrophage cell line with leishmania infantum: determination of nitric oxide production and anti-leishmanial activity

We have previously shown that resistance to Leishmania infantum in dogs is associated with a Th1 type of immune response. In this study, we use a canine macrophage cell line (030-D) that can readily be infected with this protozoan parasite. Our aim is to further characterize the effector mechanisms involved in killing of Leishmania parasite in dogs. We observed that activation of 030-D cells by incubation with a supernatant derived from a Leishmania-specific T cell line containing IFN-gamma, TNF-alpha and interleukin-2 (IL-2) resulted in enhanced nitric oxide (NO) production by these cells. In addition, we observed enhanced anti-leishmanial activity of infected 030-cells after activation. Both, NO production and anti-leishmanial activity were abrogated by addition of L-N(G)-nitroargininemethyl ester (L-NAME), an analogue of L-arginine. Thus, NO play an important role in the anti-leishmanial activity of these canine macrophages. We propose the infection of the 030-D cell line as a good in vitro model to further investigate parasite-host cell interactions in dogs, a natural host of Leishmania parasites.     

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Temporal appearance of structural and nonstructural bluetongue viral proteins in infected cells, as determined by immunofluorescence staining and flow cytometry

The temporal appearance of 4 viral proteins was detected in bluetongue virus (BTV)-infected Vero cells by indirect immunofluorescence staining with monoclonal antibodies (MAb) to BTV structural proteins VP2 and VP7 and nonstructural proteins NS1 and NS2. Bluetongue viral proteins were detected at distinct intervals after inoculation of Vero cells; VP7 was first detected 3 hours after inoculation, NS1 and NS2 at 5 hours after inoculation, whereas VP2 was not detected until 8 hours after inoculation. Patterns of fluorescence varied with the fixative used, but each MAb induced a distinct pattern of fluorescence in infected cells. Flow cytometry, which was used with each of the 4 MAb, proved to be an accurate and sensitive method of detecting BTV-infected P3 mouse myeloma cells. The temporal appearance of each viral protein in BTV-infected P3 cells was similar to that detected in BTV-infected Vero cells. Advantages of flow cytometry over conventional immunofluorescence staining to detect BTV-infected cells included: (1) enumeration of the proportion of infected cells in a population; (2) further characterization of infected cells, including estimates of their viability; and (3) computer-assisted storage and analysis of data obtained.     

Chlamydiosis in workers at a duck farm and processing plant

SUMMARY An outbreak of chlamydiosis was Investigated in workers at a duck farm and processing plant during winter 1989. Chlamydia psittaci was isolated from ducks, but there was little evidence of clinical chlamydiosis in the ducks. Serological tests showed that 76% of workers had been exposed to Infection, 12% recently, 16% had not been exposed and 8% had inconclusive test results. There was a strong association (P < 0.05) between years of employment and exposure to Infection. Infection appeared to be contracted during the first year of employment. Measures were Implemented to reduce shed contamination and control rodents and wild birds. At the same time, ducks were medicated, the processing plant was modified, work practices were altered and workers were advised about chlamydiosis to reduce the risk of human infection.     

Identification of canine T-lymphocyte subsets with monoclonal antibodies.

A panel of five murine monoclonal antibodies to canine T-lymphocytes were produced. Antibodies 4.78, 12.125 and 8.358 reacted with approximately 18%, 39% and 60% peripheral blood lymphocytes, respectively. Two color flow cytometric analysis showed that lymphocytes expressing 1.140, 4.78, 8.53 and 12.125 were subsets of lymphocytes expressing 8.358. The lymphocytes expressing 8.358 were negative for surface immunoglobulin. The subsets defined by 1.140, 4.78 or 8.53, 12.125 were mutually exclusive and together account for most cells expressing 8.358 in the peripheral blood, spleen, and lymph node. In the thymus, approximately 47% cells were positive for both 1.140/4.78 and 8.53/12.125. SDS-PAGE analysis of radiolabelled thymus cell lysates demonstrated that antibodies 1.140 and 4.78 immunoprecipitated a 32,35 kd heterodimer under reducing conditions and 12.125 immunoprecipitated a single 56 kd chain under reducing and non-reducing conditions. Antibodies 8.53/12.125 and 1.140/4.78 react with canine lymphocyte populations that occur in proportions similar to lymphocytes expressing CD4 and CD8 like molecules in several primate and non-primate species. The molecules recognized by 12.125 and 1.140/4.78 were similar in size and subunit composition to human CD4 and CD8.     

Involvement of ATP‐sensitive K+ channels in the peripheral antinociceptive effect induced by ketamine

ObjectiveTo investigate the contribution of K⁺ channels on peripheral antinociception induced by ketamine.Study designProspective experimental study.Animals110 male Wistar rats weighing 160–200 g.MethodsThe paw pressure required to elicit limb flexion was designated as the nociceptive threshold. Hyperalgesia was induced by intraplantar injection of prostaglandin E₂. All drugs were administered locally into the right hind paw of rats. Ketamine was administered into the right hind paw 2 hours and 55 minutes after local injection of PGE₂. Tetraethylammonium was administered 30 minutes prior to ketamine and the other K⁺ channel blockers, glibenclamide, dequalinium and paxilline, were administered 5 minutes prior to ketamine.ResultsProstaglandin E₂ (2 μg per paw) induced hyperalgesia. Ketamine (10, 20, 40 and 80 μg per paw) elicited a local peripheral antinociceptive effect that was antagonized by a specific blocker of ATP‐sensitive K⁺ channels, glibenclamide (20, 40 and 80 μg per paw). In another experiment, the non‐selective voltage‐dependent K⁺ channel blocker tetraethylammonium (30 μg per paw) and small and large conductance blockers of Ca²⁺‐activated K⁺ channels, dequalinium (50 μg per paw) and paxilline (20 μg per paw), were ineffective at blocking the effect of a local ketamine injection.Conclusions and clinical relevanceAnalysis of these results provides evidence that ketamine, may in part, induce peripheral antinociceptive effects by ATP‐sensitive K⁺ channel pathway activation.     

Coherent optical control of the quantum state of a single quantum Dot

Picosecond optical excitation was used to coherently control the excitation in a single quantum dot on a time scale that is short compared with the time scale for loss of quantum coherence. The excitonic wave function was manipulated by controlling the optical phase of the two-pulse sequence through timing and polarization. Wave function engineering techniques, developed in atomic and molecular systems, were used to monitor and control a nonstationary quantum mechanical state composed of a superposition of eigenstates. The results extend the concept of coherent control in semiconductors to the limit of a single quantum system in a zero-dimensional quantum dot.