Nonclinical reproductive and developmental safety evaluation of the MAGE-A3 Cancer Immunotherapeutic, a therapeutic vaccine for cancer treatment.

We assessed potential toxic effects of the MAGE-A3 Cancer Immunotherapeutic on female fertility and embryo-fetal, pre- and post-natal development in rats and on male fertility in rats and monkeys. Three groups of 48 female (Study 1) or 22 male (Study 2) CD rats received 5 or 3 injections of 100µL of saline, AS15 immunostimulant, or MAGE-A3 Cancer Immunotherapeutic (MAGE-A3 recombinant protein combined with AS15) at various timepoints pre- or post-mating. Male Cynomolgus monkeys (Study 3) received 8 injections of 500µL of saline (n=2) or the MAGE-A3 Cancer Immunotherapeutic (n=6) every 2 weeks. Rats were sacrificed on gestation day 20 or lactation day 25 (Study 1) or 9 weeks after first injection (Study 2) and monkeys, 3 days or 8 weeks after last injection. Injections were well tolerated. Female rat mating performance or fertility, pre- and post-natal survival, offspring development up to 25 days of age, and male mating performance (rats) or fertility parameters (rats and monkeys) were unaffected.

Fluoxetine-induced change in rat brain expression of brain-derived neurotrophic factor varies depending on length of treatment.

Recent studies indicate that brain-derived neurotrophic factor (BDNF) may be implicated in the clinical action of antidepressant drugs. Repeated (2-3 weeks) administration of antidepressant drugs increases BDNF gene expression. The onset of this response as well as concomitant effects on the corresponding BDNF protein is however, unclear. The present study investigated the effects of acute and chronic administration of the selective serotonin reuptake inhibitor, fluoxetine (10mg/kg p.o.), upon regional rat brain levels of BDNF mRNA and protein expression. To improve the clinical significance of the study, fluoxetine was administered orally and mRNA and protein levels were determined ex vivo using the techniques of in situ hybridisation histochemistry and immunocytochemistry respectively. Direct measurement of BDNF protein was also carried out using enzyme-linked immunosorbent assay (ELISA). Four days of once daily oral administration of fluoxetine induced decreases in BDNF mRNA (hippocampus, medial habenular and paraventricular thalamic nuclei). Whilst 7 days of treatment showed a non-significant increase in BDNF mRNA, there were marked and region-specific increases following 14 days of treatment. BDNF protein levels remained unaltered until 21 days of fluoxetine treatment, when the numbers of BDNF immunoreactive cells were increased, reaching significance in the pyramidal cell layer of CA1 and CA3 regions of Ammon's horn (CA1 and CA3) but not in the other sub-regions of the hippocampus. Indicative of the highly regional change within the hippocampus, the ELISA method failed to demonstrate significant up-regulation at 21 days, measuring levels of BDNF protein in the whole hippocampus. In contrast to the detected time dependent and biphasic response of the BDNF gene, activity-regulated, cytoskeletal-associated protein (Arc) mRNA showed a gradual increase during the 14-day course of treatment. The results presented here show that BDNF is expressed differentially depending on length of fluoxetine administration, which could contribute in explaining the slow onset of antidepressant activity observed with selective serotonin reuptake inhibitors.

Simulated intestinal fluid as transport medium in the Caco-2 cell culture model.

The Caco-2 model is widely used as a predictive tool for the oral absorption of drug candidates. Presently, transport experiments in the Caco-2 system are usually performed in 'HBSS-like' buffers. In this paper, we investigate the possibility of using simulated intestinal buffers as donor solvent during Caco-2 experiments. Toxicity assessment of these buffers on the monolayer showed that FASSIF was compatible with the Caco-2 model for at least 2 h. On the other hand, FESSIF was toxic to the monolayer. The functionality of the Caco-2 cells was assessed by determination of the transport of model compounds and the metabolic activity of hydrolases in presence of these buffers. Similar P(app) values for the (passive) theophyllin transport as well as for the (active) phenylalanine transport were obtained in TM and FASSIF. It was demonstrated that NaTC (present in FASSIF) had a P-gp inhibitory activity, as inclusion of NaTC in the apical compartment resulted in an increased absorptive and decreased secretory transport of CsA. The activity of the aminopeptidase enzyme was similar in both models. These results suggest that FASSIF can be used as an apical medium in the Caco-2 system. Since bile salts are also present in physiological conditions, the use of FASSIF may increase the relevance for the prediction of oral absorption using Caco-2 experiments.

Image analysis evaluation of ploidy, S-phase fraction and nuclear area in canine mammary tumours.

Image analysis was used on cytocentrifuge preparations of Feulgen-stained nuclei extracted from formalin-fixed, paraffin wax-embedded tissues to determine ploidy (assessed by integrated optical density), S-phase fraction, and nuclear area of 90 canine mammary tumours (30 benign and 60 malignant). Only two lesions identified histologically as benign were aneuploid, suggesting potential malignancy. Of malignant tumours, 37% were aneuploid. No distinction based on ploidy could be made within benign or malignant groups, except for tubular adenocarcinomas of complex type, all of which were diploid. Mean S-phase fraction of malignant lesions (15.60%) was twice that of benign lesions (7.87%; P < 0.0001). The mean nuclear surface was significantly smaller in malignant lesions (70.62 microns2) than in benign counterparts (77.45 microns2; P < 0.05). No relation was found between a given tumour type and S-phase fraction or nuclear area within histologically malignant or benign tumour groups. Infiltration of lymphatics by neoplastic cells, known to indicate a poor prognosis, was associated with aneuploid lesions (mean DNA index > 1.25) and high S-phase fraction. The presence of necrosis and of a high number of mitoses was significantly associated with high S-phase fraction lesions.

Comparison of argyrophilic nucleolar organizer regions by counting and image analysis in canine mammary tumors.

Two techniques for evaluating argyrophilic nucleolar organizer regions (AgNOR) were compared on 74 canine mammary tumors to discriminate between benign and malignant lesions. For each lesion, direct counting of AgNOR on at least 100 cell nuclei was compared with area, perimeter, and integrated optical density AgNOR dot values determined by image analysis. Significant differences between benign and malignant tumors were observed with both methods; however, lesions determined as aggressive or proliferative by histologic evaluation were only singled out by image analysis measurements. Image analysis, in our hands, was a reliable, precise, and convenient technique to characterize malignancy in canine mammary tumors.

Immunohistochemical identification of myoepithelial, epithelial, and connective tissue cells in canine mammary tumors.

Fifty-eight formalin-fixed paraffin-embedded canine mammary tumors, 19 malignant and 39 benign, were used in this study. Tumors were obtained from dogs submitted for surgical resection of lesions at private veterinary practices in Brussels or from the surgery unit of the Faculty of Veterinary Medicine, University of Liège. Immunohistochemical evaluation was performed, using monoclonal antibodies directed against keratins 8-18 and 19, vimentin, desmin, and alpha-actin and polyclonal antibodies directed against high-molecular-weight keratins and S-100 protein. The main cell types, epithelial, myoepithelial, and connective, were identified, and myoepithelial cells represented the major component of most tumors, both benign and malignant. Myoepithelial cells had five patterns: resting and proliferative suprabasal cells, spindle and star-shaped interstitial cells, and cartilage. Reactivity to keratin 19, vimentin, alpha-actin, and S-100 protein suggested a progressive transformation from resting cells to cartilage. Epithelial cell reactivities were limited to keratins; only keratinized cells were positive for polyclonal keratins. Myofibroblasts were positive for both vimentin and alpha-actin, and connective tissue cells were positive for vimentin. Myoepithelial cells appeared to be the major component of carcinomas, justifying reevaluation and simplification of histomorphologic classifications, with a "pleomorphic carcinoma" group including all carcinomas except squamous, mucinous, and comedo carcinomas. Immunohistochemical evaluation, in addition to routine hematoxylin and eosin histopathologic evaluation is recommended for precise classification of canine mammary tumors.