Multinucleated giant cells with an osteoclast phenotype derived from caprine peripheral blood mononuclear cells

Formation of multinucleated giant cells (MGCs) by macrophage fusion is a typical cytopathic effect of lentiviral replication in caprine monocytes and MGC formation from cultured caprine peripheral blood mononuclear cells (PBMCs) has been considered to be diagnostic for small ruminant lentivirus (SRLV) infection. In this study, formation of MGCs was observed after 7–14 days when PBMCs were cultured from healthy goats free from SRLV infection. These MGCs expressed tartrate-resistant acid phosphatase, calcitonin receptor, integrin αVβ3, cathepsin K and matrix metalloproteinase 9 and were able to resorb bone in vitro in the absence of RANKL and macrophage colony stimulating factor, consistent with an osteoclast phenotype.     

Canine corneal fibroblast and myofibroblast transduction with AAV5

Objective The aims of this study were (1) to determine the efficacy of adeno‐associated vector serotype 5 (AAV5) for delivering gene therapy to canine corneal fibroblasts (CCFs) and myofibroblasts (CCMs) using enhanced green fluorescent protein (GFP) marker gene and (2) to evaluate the cytotoxicity of AAV5 to CCFs and CCMs using an in vitro model. Methods Healthy donor canine corneas were used to generate primary CCFs by growing cultures in minimal essential medium supplemented with 10% fetal bovine serum. Canine corneal myofibroblasts were produced by growing cultures in serum‐free medium containing transforming growth factor β1 (1 ng/mL). An AAV5 titer (6.5 × 10¹² μg/mL) expressing GFP under control of hybrid cytomegalovirus + chicken β‐actin promoters (AAV5‐gfp) was used to transduce CCF and CCM cultures. Delivered gene expression in CCFs and CCMs was quantified using immunocytochemistry, fluorescent microscopy, and real‐time PCR. Transduction efficacy of the AAV5 vector was determined by counting DAPI‐stained nuclei and EGFP‐positive cells in culture. Phase‐contrast microscopy, trypan blue, and dUTP nick end labeling (TUNEL) assays were used to determine the toxicity and safety of AAV5 in this canine corneal model. Results Topical AAV5 application successfully transduced a significant population of CCFs (42.8%; P < 0.01) and CCMs (28%; P < 0.01). Tested AAV5 did not affect CCF or CCM phenotype or cellular viability and did not cause significant cell death. Conclusions The tested AAV5 is an effective and safe vector for canine corneal gene therapy in this in vitro model. In vivo studies are warranted.     

Pharmacokinetics of intravenous ketorolac in cats undergoing gonadectomy

AIM: To determine the pharmacokinetics of ketorolac tromethamine (0.5?mg/kg) when administered I/V to cats undergoing gonadectomy.

METHODS: Ketorolac was administered to nine female and three male shorthair domestic cats as an I/V bolus of 0.5?mg/kg after intubation, and 20 minutes prior to ovariectomy or orchiectomy. Intra-operative cardiorespiratory variables were monitored and blood samples were collected over 24 hours. Concentrations of ketorolac in serum were determined by high-performance liquid chromatography to establish pharmacokinetic parameters.

RESULTS: During surgery, mean end tidal isoflurane concentration was 1.63 (SD 0.24)% and normocapnia and spontaneous ventilation were maintained in all animals. The kinetics of ketorolac was described by a two-compartment model. The distribution and elimination half-lives were 0.09 (SD 0.06) and 4.14 (SD 1.18) hours, respectively. The body clearance was 56.8 (SD 33.1) mL/h/kg. The volume of distribution at steady-state and the mean residence time were 323.9 (SD 115.7) mL/kg and 6.47 (SD 2.86) hours, respectively.

CONCLUSION AND CLINICAL RELEVANCE: On the basis of the results, concentrations of ketorolac in serum in cats were above the human effective concentrations for 5–6 hours postoperatively. However, other studies including a control group are advocated to further investigate the ketorolac kinetics and the analgesic efficacy in this species.     

Ecotoxicological evaluation of Harbour sediments using marine organisms from different trophic levels

Background, Aim and Scope  The toxicity of contaminated sediments should be evaluated considering the direct exposure of laboratory organisms to whole sediments and the indirect exposure to elutriates or extracts (Tay et al. 1992, Byrne and Halloran 1999, Nendza 2002). The alga Dunaliella tertiolecta is indicated for the use in toxicity bioassays because it is highly sensitive to several xenobiotics. Harpacticoid copepods have been already used for toxicity testing and Tigriopus fulvus is a promising Mediterranean target-species in ecotoxicology (Todaro et al. 2001, Faraponova et al. 2003, Pane et al. 2005a). In this study, the toxicity of sediments collected in harbour sites of the Northeastern Adriatic Sea was evaluated by growth inhibition test with free living and alginate-immobilized Dunaliella tertiolecta and acute toxicity test with nauplii and adult Tigriopus fulvus with the aim of pointing out the importance to utilize model organisms from different trophic levels in sediment ecotoxicology. Methodology  Elutriates and whole sediments were tested on free living and immobilized (Pane et al. 1998) algal cells, and on laboratory reared copepods. Free-living D. tertiolecta were exposed to diluted elutriates in a static, multi-well plate system. Naalginate immobilized D. tertiolecta were placed in polystyrene inserts fitted with polyester mesh bottoms and exposed to a thin layer (2 mm) of whole sediments in multi-well plates (EPS 1992, Pane and Bertino 1999). Toxicity tests with copepods were carried out on Tigriopus fulvus nauplii (elutriates) and adults (whole sediments and elutriates). Same-aged nauplii useful for toxicity tests were obtained by egg sac detaching and consequent hatching stimulation (Pane et al. 2006). Newborn nauplii (I–II stage) were exposed to elutriates in multi-well plates provided with polystyrene inserts. Adult T. fulvus maintained in polystyrene inserts fitted with polyester mesh bottoms were placed in contact with a thin layer (2 mm) of whole sediment placed on multi-well plate bottoms. All end-points were evaluated after 96 h. Results  In general, the effects increased with the increasing of elutriate concentration up to 50%; the stimulation or inhibition of algal growth was statistically significant in comparison to the control. The inhibiting elutriates induced EC₅₀ variations of algal growth ranging from 66.9% to 74.3%. The mortality of T. fulvus nauplii was always < 25% after treatment with 100% elutriates and < 10% after treatment with 50% dilution; no effect was shown up with 25% dilution; therefore LC₅₀ was not calculable. The effect of elutriates was negligible on adult copepods and LC₅₀ values were never calculable; percent mortality always resulted in < 10% after treatment with whole sediments. Discussion  Both experimental systems gave substantially similar results after exposition to whole sediments and elutriates. During the experiment with algal cells, the immobilization in Na-alginate and the employment of inserts which allowed the contact of organisms with sediments and their easy counting were particularly useful. Likewise, the employment of inserts of adequate mesh size in the tests with copepods allowed the contact of organisms with the sediment and made organism handling and counting easy, as well as the evaluation of mortality. The methodology here described and the utilization of the proposed test-species could have an importance also considering that the current trend in ecotoxicological research is towards finding the most appropriate organism for specific areas of concern by using indigenous species (Mariani et al. 2006) and towards the major significance of chronic and reproductive end-points. Conclusions  Based on the above results, it can be stated that the bioassay with Dunaliella tertiolecta could be a good estimation tool for the ecotoxicological assessment of marine sediments. The immobilization of algae in Na-alginate was seen to be useful to evaluate the toxicity of whole sediments; the employment of polystyrene inserts allowed an improvement of the procedures. T. fulvus nauplii and adults, as other harpacticoids such as Tigriopus japonicus (Yoon et al. 2006), satisfy the basic criteria for the employment of a standard species in marine bioassays. To date only pelagic Acartia tonsa are utilized in the standardized procedure to evaluate the risk assessment of chemicals or wastewaters (ISO 1999). As, on the contrary, the exposure of copepods to solid-phase contaminants it is not yet standardized, the employment of polystyrene inserts improved the procedures for T. fulvus too. So, the rapidity and the possibility to solve practical problems could be the main attractive features of this technique (Pane et al. 2005a) when applied to whole sediments. Recommendations and Perspectives  The methodology here developed being also applicable to long term and reproduction tests should be recommended because it provides relevant information in comparison with other frequently applied, standardized biotests with crustaceans (ISO 1999). The procedure has been shown to be easily applicable to selected marine organisms. ESS-Submission Editor: Prof. Dr. Henner Hollert (henner.hollert@bio5.rwth-aachen.de)     

Local photodynamic therapy for equine squamous cell carcinoma: evaluation of a novel treatment method in a murine model

The objective of this study was to evaluate the effect of local photodynamic therapy (PDT) with verteporfin on tumor growth inhibition of squamous cell carcinoma (SCC) in a murine model. SCC was implanted in 85 nude mice by subcutaneous injection of A-431 SCC cells. Treatment groups (10 mice/group) received an intra-tumoral injection of verteporfin dissolved in dimethyl sulfoxide (DMSO) or 5% dextrose solution at a dose of 0.01 or 0.1mg/cm3. Controls received only solvent, or no injectate. All groups received identical light illumination (100J/cm2). Relative change in tumor volume (RCTV) at day 30 was compared between groups using the Wilcoxon rank sum test (P< 0.05). Local PDT with verteporfin at a dose of 0.1mg/cm3 resulted in significantly lower RCTV at day 30 compared to controls. Choice of solvent (DMSO versus D5W) did not affect the results. Local PDT may be an effective adjunctive therapy for the treatment of periocular equine SCC.     

Photodynamic therapy for the treatment of periocular squamous cell carcinoma in horses: a pilot study

Objective  Local photodynamic therapy (PDT) is a novel cancer therapy in veterinary ophthalmology. A prospective pilot study seeking to demonstrate proof of principle and safety for the treatment of equine periocular squamous cell carcinoma (PSCC) was therefore conducted. We hypothesized that surgical excision with adjunctive local PDT is an effective and safe treatment for equine PSCC.
Procedures  Nine horses (10 eyes) with PSCC were treated with surgical resection, local infiltration of resulting wound beds with 2-[1-hexyloxyethyl]-2-devinylpyropheophorbide-a (HPPH) and irradiation with 665-nm wavelength diode laser. Regular follow-up ophthalmic examinations were performed.
Results  Surgical resection and PDT yielded disease-free intervals of 25–68 months in our study horses as of January, 2008. These results were obtained following a single treatment in seven horses and two treatments in one horse. In one horse, carcinoma in situ developed 2.5 months after partial surgical excision and PDT, requiring local excision under standing sedation.
Conclusions  Preliminary results suggest that surgical resection and adjunctive local PDT is a safe and effective novel treatment for PSCC in horses. More research is needed before PDT for the treatment of equine PSCC can be adequately compared with other current modalities. Important to future investigations regarding PDT, tumor recurrence rate, length of hospitalization, number of treatment episodes required to effect tumor remission, and total treatment costs should be examined in a controlled manner. Our present results and experiences suggest that this treatment may be useful in the treatment of equine PSCC.     

In vitro susceptibility patterns of Aspergillus and Fusarium species isolated from equine ulcerative keratomycosis cases in the midwestern and southern United States with inclusion of the new antifungal agent voriconazole

Objective  To evaluate and compare the in vitro susceptibility of Aspergillus and Fusarium spp. isolated from horses with ulcerative keratomycosis, address regional differences in equine keratomycosis isolates, and provide susceptibility data to update prior studies.
Animal studied  Fourteen horses with ulcerative keratomycosis.
Procedures  Banked fungal isolates from equine corneal ulcers (eight Aspergillus spp. and six Fusarium spp.) were identified at The University of Texas Health Science Center at San Antonio. In vitro minimum inhibitory concentration and susceptibility to natamycin, fluconazole, itraconazole, voriconazole, ketoconazole, and miconazole were determined for each isolate.
Results  Fungi were significantly more susceptible to voriconazole than to natamycin, itraconazole, fluconazole, and ketoconazole, but miconazole susceptibility did not differ significantly from voriconazole. Aspergillus spp. were most susceptible to voriconazole, miconazole, and itraconazole, which were significantly better to fluconazole and ketoconazole. Fusarium spp. susceptibility was greatest to natamycin and voriconazole and lowest to itraconazole and ketoconazole. Fusarium spp. were significantly less susceptible to itraconazole and ketoconazole compared to natamycin. No significant differences in susceptibility were found when isolates from Florida were compared with isolates from other states.
Conclusions and clinical relevance  Based on in vitro evidence, voriconazole appears to be the most effective antifungal for initial treatment of equine keratomycosis in the midwestern and southern United States. Results are comparable with previous studies in that isolated fungi from equine keratomycosis cases showed consistently poor susceptibility to fluconazole. Organisms isolated in different geographic locations of the midwestern and southern United States appeared to have similar patterns of antifungal susceptibility.     

CNS and lung metastasis of sebaceous epithelioma in a dog

Sebaceous epithelioma is a common canine cutaneous neoplasm characterized by a preponderance of basaloid cells with few well-differentiated sebocytes. It is considered a low-grade malignancy, as it may occasionally recur at the excision site; the possibility of lymph node metastasis is anecdotally reported, and distant metastases have never been observed. This case report presents the clinical and pathological features of a sebaceous epithelioma of the upper lip with a highly aggressive behaviour. The patient was a 9-year-old female dachshund that developed local recurrence 11 months after the excision of the primary sebaceous epithelioma and multiple lung and central nervous system metastases 5 months later. The designation epitheliomatous sebaceous carcinoma has been suggested for aggressive sebaceous epitheliomas, although differential criteria are still to be determined.     

The effects of two different light intensities on the production and behavioural traits of Italian heavy pigs

Eighty Italian heavy pigs having an initial body weight of about 75 kg were allotted to two experimental groups: a low light intensity group (LL), in which pigs received the lowest level of illumination mandatory in piggeries (40 lux) and a high light intensity group (HL), in which pigs were exposed to a higher level of illumination (80 lux). For both groups the light phase lasted 12 hours per day. Pigs were slaughtered at about 160 kg body weight. Light intensity showed to have no effects on the growth traits, carcass parameters or neutrophil-to-lymphocyte ratio of heavy. With the exception of drinking (more frequently observed among LL pigs; P < 0.01) and sternal recumbency (more frequently observed among pigs belonging to HL group; P < 0.01), the behavioural observations, carried out on 20 pigs per group, indicated that light intensity did not affect the main behavioural traits of pigs. Agonistic interactions were significantly higher (P < 0.05) among pigs receiving a lower level of illumination (40 lux; LL group) and pigs exposed to the higher level of illumination (80 lux; HL group) showed a tendency (P < 0.1) towards a higher proportion of tactile social interactions. Our results confirm that a proper illumination level can be included among environmental conditions having a positive effect in order to preserve swine welfare.