Mouse bioassay for in vivo screening of oestrogen and progesterone antagonists

This study tested and compared the anti-proliferative and proliferative activities of two anti-oestrogens and three anti-progestins on four separate mouse model systems: young intact and adult ovariectomized (OV-X) females, and young intact and adult castrated males. Pure steroidal anti-oestrogen ICI 182,780 (ICI) decreased mammary and uterine growth stimulated by endogenous hormones in young intact females and by exogenous hormones [progesterone (Prog), 17beta-oestradiol (E) or E plus Prog] in both young intact and adult ovariectomized (OV-X) females. Non-steroidal anti-oestrogen EM-800 (EM), on the other hand, had no effect on mammary and uterine growth stimulated by endogenous hormones in young intact females and in adult OV-X females. Uterine growth was even stimulated by EM alone, and a combination of EM plus Prog not only stimulated uterine growth but also mammary growth (an oestrogenic agonistic activity). However, EM showed anti-oestrogenic activities in both mammary and uterine tissues in females treated with E or E plus Prog. In males, ICI and EM decreased mammary growth stimulated by exogenous hormones (E or E plus Prog) in both young intact and adult castrated animals. In young intact, but not in adult castrated males, ICI increased seminal vesicle growth affected by both endogenous and exogenous (Prog, E or E plus Prog) hormones. EM, on the other hand, decreased seminal vesicle weights in E or E plus Prog and increased its weights in Prog-treated young intact males. Thus, under certain conditions EM possess mixed agonist and antagonist activity in the mammary gland, uterus and seminal vesicles. Norethindrone acetate (NA)-stimulated mammary growth was decreased by anti-progestins onapristone (ON), RU 46556 (RU), and RU 38486 (MI) by 34-59% in females and by 35-93% in males. Uterine weights of NA-treated females were decreased by ON and RU by 29-55% but not by MI. In NA-treated young intact males, seminal vesicle weights were stimulated by RU (by 63%) and not affected by ON and MI. In NA-treated adult castrated males, seminal vesicle weights were decreased by ON, increased by RU and not affected by MI. The results obtained in these and our earlier studies show clearly that mouse four-model systems could serve as in vivo tool for the detection of steroid hormone agonist and antagonist activities of natural and man-made chemicals.     

Mouse hepatoblastomas: a histologic, ultrastructural, and immunohistochemical study

Hepatoblastomas from B6C3F1 and BALB/c mice were examined by light and electron microscopy and by immunohistochemical reactions for alpha-fetoprotein, keratin, and vimentin. Tumors occurred in one group of a chronic bioassay for the interaction of diet, genetic strain, and the carcinogen, 2-acetylaminofluorene. Tumors had several populations (including epithelial and mesenchymal cells) in various stages of differentiation. Neoplastic epithelial cells had features of embryonal hepatocytes, such as sparse cytoplasmic organelles, absence of glycogen, abundant free ribosomes, occasional bile canaliculi, and peroxisome-like dense bodies. Embryonal fibroblast-like cells had pleomorphic and folded nuclei with prominent perinuclear chromatin and dispersed cytoplasmic organelles. Fibroblast-like cells were surrounded by bundles of collagen fibrils. Intermediate or transitional types of cells were seen. No tumor cells were immunoreactive for mouse alpha-fetoprotein (AFP) antibody, unlike those in hepatocellular adenomas or carcinomas. Epithelial and mesenchymal tumor cells contained intermediate filaments throughout the cytoplasm; some of these cells stained for keratin but not for vimentin. These findings suggest that mouse hepatoblastomas are derived from bipotential liver blastema cells and are composed of a mixture of several cell populations.     

The effect of zeranol and testosterone on Merino wethers exposed to highly oestrogenic subterranean clover pasture

Groups of Merino wethers treated with 2 doses of zeranol (6 mg and 12 mg), or testosterone cyclopentyl propionate (150 mg) and untreated controls were grazed at 2 sites, one an oestrogenic subterranean clover (Trifolium subterraneum) pasture and the other a low oestrogen medic (Medicago truncatula) pasture. The influence of oestrogenic subterranean clover on these treatments was assessed by measuring changes in teat length, bulbourethral gland weight and pathology, bodyweight, carcase weight, dressing percentage and greasy and clean fleece weights. Teat lengths were increased by all treatments except 6 mg of zeranol where increases were not significant, and although increased by exposure to oestrogenic pasture this effect were not additive. Bulbourethral gland weights were increased by both of the zeranol treatments and by oestrogenic pasture, and these effects appeared to be additive. Differences observed histologically indicated that testosterone protected whereas zeranol exacerbated the influence of oestrogen. The bodyweights of all treated groups were heavier than the controls, but carcase weights were not significantly different. However an effect was seen in the group given the 6 mg dose of zeranol on the low oestrogen site, where the dressing percentage was significantly lower than in the control and testosterone treated groups. Differences in greasy and clean fleece weights were not significant except that the washing yield of the testosterone-treated group was significantly lower at the low oestrogen site.     

Cloning, expression and bioassay of canine CTLA4Ig

Blockade of the B7:CD28 costimulatory pathway has been shown to inhibit humoral immunity, graft rejection, graft versus host disease and ameliorate autoimmune diseases. A soluble chimeric fusion protein, CTLA4Ig, binds to B7 with greater affinity than CD28 and blocks the binding of CD28 to B7. We describe the cloning and expression of canine CTLA4Ig, a recombinant chimeric fusion protein composed of the extracellular domain of canine CTLA-4 and the CH2-CH3 domains of canine immunoglobulin alpha constant region (IGHA) genes, linked via an immunologically inert flexible peptide. The recombinant CTLA4Ig protein of approximately 45kDa molecular weight was expressed mainly as insoluble inclusion bodies in Escherichia coli. The protein was solubilized in denaturing buffer and purified using nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatography followed by refolding. The yield was about 6mg of recombinant CTLA4Ig per liter of culture. The purified protein was biologically active in one-way mixed lymphocyte reactions, demonstrating immunosuppressive activities in a dose-dependent manner. The findings suggest that recombinant canine CTLA4Ig protein could be valuable in assessing the function of CTLA-4 in the canine immune system and may be effective in autoimmune disease therapy.     

Determination of excretion of inulin, creatinine, sodium sulfanilate, and phenolsulfonphthalein to assess renal function in goats

Excretion of creatinine, sodium sulfanilate (SS), and phenolsulfonphthalein (PSP) was studied in healthy goats. In conscious goats, mean (+/- SEM) inulin clearance was 2.26 +/- 0.08 ml/min/kg of body weight. Endogenous creatinine clearance, 1.97 +/- 0.09 ml/min/kg, underestimated inulin clearance (P less than 0.01), probably because of the presence of noncreatinine chromogens in caprine plasma. The estimated renal clearance of PSP was 6.88 +/- 0.39 ml/min/kg, whereas the estimated renal clearance of SS was 3.71 +/- 0.39 ml/min/kg. Both exceeded inulin clearance (P less than 0.01), confirming renal tubular secretion of both compounds. In 6 anesthetized goats, exogenous creatinine clearance and SS clearance exceeded inulin clearance (P less than 0.05). Results of stop-flow experiments documented secretion of creatinine and SS by the proximal portion of the caprine nephron. Plasma half-life of PSP in uninephrectomized goats exceeded that in intact goats (20.2 +/- 1.5 min vs 11.9 +/- 0.7 min; P less than 0.01). Similarly, plasma half-life of SS was greater in goats after uninephrectomy (58.2 +/- 6.2 min vs 30.4 +/- 1.2 min; P less than 0.01).     

New approach to assess sperm DNA fragmentation dynamics: Fine-tuning mathematical models

Background: Sperm DNA fragmentation(sDF) has been proved to be an important parameter in order to predict in vitro the potential fertility of a semen sample. Colloid centrifugation could be a suitable technique to select those donkey sperm more resistant to DNA fragmentation after thawing. Previous studies have shown that to elucidate the latent damage of the DNA molecule, sDF should be assessed dynamically, where the rate of fragmentation between treatments indicates how resistant the DNA is to iatrogenic damage. The rate of fragmentation is calculated using the slope of a linear regression equation. However, it has not been studied if s DF dynamics fit this model. The objectives of this study were to evaluate the effect of different after-thawing centrifugation protocols on sperm DNA fragmentation and elucidate the most accurate mathematical model(linear regression, exponential or polynomial) for DNA fragmentation over time in frozen-thawed donkey semen.Results: After submitting post-thaw semen samples to no centrifugation(UDC), sperm washing(SW) or single layer centrifugation(SLC) protocols, sD F values after 6 h of incubation were significantly lower in SLC samples than in SW or UDC.Coefficient of determination(R~2) values were significantly higher for a second order polynomial model than for linear or exponential. The highest values for acceleration of fragmentation(aSDF) were obtained for SW, fol owed by SLC and UDC.Conclusion: SLC after thawing seems to preserve longer DNA longevity in comparison to UDC and SW. Moreover,the fine-tuning of models has shown that sDF dynamics in frozen-thawed donkey semen fit a second order polynomial model, which implies that fragmentation rate is not constant and fragmentation acceleration must be taken into account to elucidate hidden damage in the DNA molecule.     

Muscovy ducklings, a particularly sensitive avian bioassay for T-2 toxin and diacetoxyscirpenol

Day-old domesticated Muscovy ducklings (Cairina moshata) were given the trichothecene mycotoxins T-2 toxin or diacetoxyscirpenol at 0.25, 0.5, or 1 micrograms/g of feed for 7 days. All concentrations of both toxins caused characteristic dose-related lesions on the roof of the oral cavity of the birds. Muscovy ducklings showed these signs in a shorter time (less than 16 hours in some cases) and at a lower feed concentration of these trichothecenes than did other avian species in similar studies.     

小鼠肝实质细胞的分离、鉴定及原代培养

为分离、培养高纯度原代小鼠肝实质细胞,并鉴定其纯度及生物活性,试验采用原位两步循环灌流法及多次低速差速离心法分离纯化肝实质细胞,促贴壁培养基原代培养,台盼蓝检测其存活率,PAS反应检测其糖原合成能力,免疫荧光化学检测细胞中CK18表达情况。结果表明:每只小鼠可获取约1.5×10^6个细胞,存活率>97%;镜下发现细胞在接种后6h开始贴壁,72h贴壁细胞生长状态良好,胞体变大、不规则,细胞间相互靠拢呈岛状或条索状连接;肝细胞出现成片紫红色糖原颗粒,CK18在细胞中均匀分布;糖原反应联合CK18免疫荧光显示细胞纯度在90%以上。说明该试验所用方法分离出肝实质细胞数量和存活率高,促贴壁培养基培养的肝实质细胞纯度高,为细胞代谢、细胞毒性等的研究奠定了基础。     

A Swiss case-control study to assess Neospora caninum-associated bovine abortions by PCR, histopathology and serology.

Neospora caninum is one of the most frequent infectious organisms causing abortion in cattle worldwide. The present case-control study was designed to assess the importance of bovine neosporosis for causing abortion in Swiss cattle and to identify selected risk factors. Infection was primarily diagnosed by a N. caninum-specific PCR and serology, complemented with histopathology and immunohistochemistry. A total of 113 case and 113 corresponding control-farms were studied for 1.5 year. During this time period, 242 abortions were reported and referred for bacteriological, virological, parasitological and pathohistological examinations. N. caninum was detected by PCR in the brains of 21% of all aborted fetuses. Microscopic lesions indicative for cerebral protozoa infection were detected in 84% of PCR-positive fetal brains. Bovine viral diarrhea virus (BVDV) was demonstrated in 7% of the cases, and bacterial infections were detected in 4% of the abortions. One or more N. caninum-abortions occurred in 20% of the herds (41 case-farms and 3 control-farms). Serological examination of aborting mother cows revealed a significantly higher percentage of N. caninum-seropositive animals (44%) in comparison to the prevalence in a randomly selected population (12%). However, in eight cases (4% of all investigated abortions) seronegative cows aborted N. caninum PCR-positive fetuses, and in 50 cases the fetus remained negative although the respective mother cow was N. caninum-seropositive. Repetitive serological investigations (at a 3-12 months interval) of 3551 cows from case- and control-farms showed a decrease of the overall N. caninum-seroprevalence from 17 to 12%. Ninety out of 3008 seronegative animals were converted to N. caninum-seropositivity. Conversely, 212 out of 543 initially seropositive animals became seronegative for their second serum sample. The obtained data underlined the importance of N. caninum as a causative agent for abortion in Swiss cattle. Furthermore, PCR was confirmed to be a valuable diagnostic tool for the primary diagnosis of N. caninum in aborted fetuses. On the other hand, the value of serology appears to be hampered by the temporal instability of N. caninum antibody concentrations in adult cattle, including especially seronegativity of some individual animals. Thus, seronegativity in a mother cow or heifer does not exclude N. caninum-associated abortions.     

Application of diethylstilbestrol dipropionate in bulls. II. Residues in bile, liver, kidney, muscle tissues and application site

The second part of an experiment is described in which 20 one year old bulls were injected with diethylstilbestrol (DES) dipropionate containing preparations. Analysis of DES content was performed in several tissues, such as the injection site, diaphragm muscle, psoas muscle, liver, kidney and bile. In the injection site appreciable amounts of DES were found. Measurable amounts of DES were also found in liver and kidney until 4 weeks after injection. In bile, DES concentrations were even higher than those in urine, and were well correlated with DES concentrations in urine. Implications for screening purposes are discussed.     

Functional MRI as a tool to assess vision in dogs: the optimal anesthetic

Functional magnetic resonance imaging (fMRI) is a recent advance in neuroimaging that provides a picture of brain activity with excellent spatial resolution. Current methods used to evaluate canine vision are poorly standardized and vulnerable to bias. Functional MRI may represent a valuable method of testing vision in dogs if the impacts of anesthesia on fMRI are understood. Six dogs were scanned during visual stimulation, each under three different anesthetic protocols (isoflurane, propofol, fentanyl/midazolam) to address the questions: (1) Can visually evoked fMR signals be reliably recorded in anesthetized dogs? and (2) Which anesthetic agent permits the least suppression of visually induced fMR signal in dogs? This study confirms that visual stimuli reliably elicit neural activity and fMR signal change in anesthetized dogs. No significant differences in images acquired under the three anesthetics were found, and there was no significant relationship between anesthetic dose and brain activity, within the range of doses used in this study. Images obtained during isoflurane anesthesia were more consistent between dogs than those obtained with the other two agents. This reduced variation may reflect the fact that inhalant anesthesia is more easily controlled than intravenous anesthesia under conditions associated with high field fMRI.     

Use of client-specific outcome measures to assess treatment effects in geriatric, arthritic dogs: controlled clinical evaluation of a nutraceutical

A questionnaire method was designed for dog owners to monitor the orthopedic disabilities of their pets for evaluation of a nutraceutical with joint health claims. Fifty large-breed dogs, 7 to 12 years of age, presenting with signs of osteoarthritis, were randomly allocated to placebo and active treatment groups. Degree of disability was assessed by physical examination, a standard questionnaire on daily activities, and a case-specific questionnaire that monitored specific impairments of each dog. The test product was a special milk protein concentrate (SMPC) from hyperimmunized cows, previously shown to express antiinflammatory and antiarthritic activity in humans. After a 1-week run-in period of dosing with placebo, each dog was randomly assigned to a treatment and given gelatin capsules containing either SMPC or a placebo twice daily for 8 weeks. Overall improvement was noted in 68% and 35% of the SMPC and placebo groups, respectively. Significant (P <.05) improvement in mean standardized and patient-specific questionnaire scores and in owner global assessments was detected in the SMPC group but not in the placebo group. Compared with the placebo group, the treatment response was significantly better in the SMPC group with regard to case-specific scores (P lt;.001) and owner global assessments (P =.004). The product was well tolerated and serum chemistry findings remained within normal limits.     

Use of client-specific outcome measures to assess treatment effects in geriatric,arthritic dogs: controlled clinical evaluation of a nutraceutical

A questionnaire method was designed for dog owners to monitor the orthopedic disabilities of their pets for evaluation of a nutraceutical with joint health claims. Fifty large-breed dogs, 7 to 12 years of age, presenting with signs of osteoarthritis, were randomly allocated to placebo and active treatment groups. Degree of disability was assessed by physical examination, a standard questionnaire on daily activities, and a case-specific questionnaire that monitored specific impairments of each dog. The test product was a special milk protein concentrate (SMPC) from hyperimmunized cows, previously shown to express antiinflammatory and antiarthritic activity in humans. After a 1-week run-in period of dosing with placebo, each dog was randomly assigned to a treatment and given gelatin capsules containing either SMPC or a placebo twice daily for 8 weeks. Overall improvement was noted in 68% and 35% of the SMPC and placebo groups, respectively. Significant (P <.05) improvement in mean standardized and patient- specific questionnaire scores and in owner global assessments was detected in the SMPC group but not in the placebo group. Compared with the placebo group, the treatment response was significantly better in the SMPC group with regard to case-specific scores (P <.001) and owner global assessments (P =.004). The product was well tolerated and serum chemistry findings remained within normal limits.