Kenneth J. Snibson

Biomedical applications of sheep models: from asthma to vaccines.

Although rodent models are very popular for scientific studies, it is becoming more evident that large animal models can provide unique opportunities for biomedical research. Sheep are docile in nature and large in size, which facilitates surgical manipulation, and their physiology is similar to humans. As a result, for decades they have been chosen for several models and continue to be used to study an ever-increasing array of applications. Despite this, their full potential has not been exploited. Here, we review the use of sheep as an animal model for human vaccine development, asthma pathogenesis and treatment, the study of neonatal development, and the optimization of drug delivery and surgical techniques.

Airway remodelling and inflammation in sheep lungs after chronic airway challenge with house dust mite

Background Remodelling of airway walls is a significant morbidity factor in patients suffering from chronic asthma. The relationship between airway remodelling and the inflammatory response is not well defined. Sheep have been used extensively to model airway disease in humans and represent a suitable model to examine airway remodelling.

Induction of allergic inflammation in the lungs of sensitized sheep after local challenge with house dust mite

Background Previous sheep models of asthma are based on sheep sensitized to nematode (Ascaris) allergens and these have been used to evaluate the physiological and pharmacological effects of potential anti‐asthma agents. The immunological mechanisms associated with the allergic response in sheep lungs has not been examined in detail.

Pulmonary delivery of ISCOMATRIX influenza vaccine induces both systemic and mucosal immunity with antigen dose sparing.

Using a large animal model, we evaluated whether delivery of influenza vaccine via its mucosal site of infection could improve vaccine effectiveness. Unexpectedly, pulmonary immunization with extremely low antigen doses (0.04 μg influenza) induced serum antibody levels equivalent to those resulting from a current human vaccine equivalent (15 μg unadjuvanted influenza, subcutaneously) and vastly superior lung mucosal antibodies. Induction of this potent response following lung vaccination was dependent on addition of ISCOMATRIX adjuvant and deep lung delivery. Functional antibody activity, marked by hemagglutination inhibition, was only present in the lungs of animals that received adjuvanted vaccine via the lungs, suggesting this approach could potentially translate to improved protection. The 375-fold reduction in antigen dose and improved mucosal antibody responses, compared to the current vaccine, suggests that mucosal delivery via the pulmonary route may be particularly relevant in the event of an influenza pandemic, when vaccine supplies are unlikely to meet demand.

Combined mucosal and systemic immunity following pulmonary delivery of ISCOMATRIX adjuvanted recombinant antigens.

Deep pulmonary delivery of an influenza ISCOMATRIX vaccine has previously been shown to induce a combined mucosal and systemic antibody response. To explore whether this combined response is influenced by intrinsic properties of the component antigen, we examined the efficacy of deep pulmonary delivery of ISCOMATRIX vaccines containing different recombinant antigens, specifically gB glycoprotein from cytomegalovirus and a fragment of catalase from Helicobacter pylori. Both these vaccines induced antigen-specific mucosal and systemic immunity, as well as antigen-specific proliferative cellular responses. Pulmonary immunisation with ISCOMATRIX vaccines may therefore be a generic way of inducing combined systemic and mucosal immunity.

Ultrastructural localization and gradient of activity of alkaline phosphatase activity during rodent odontogenesis

SummaryThe ultrastructural localization and gradient of activity of alkaline phosphatase were studied with respect to cell differentiation, matrix synthesis, and matrix mineralization in the incisor and molar teeth of 4-day-old Sprague-Dawley rats. The animals were perfused intracardially at room temperature with 2.5% glutaraldehyde in 0.1M sodium cacodylate (pH 7.4) with 3–4% sucrose. The jaws were dissected, immersion-fixed for 24 h, and the incisor and molar tooth germs removed. These were demineralized in 10% EDTA in NaOH (pH 7.4) with 7% sucrose. After reactivation of the enzyme with 0.1M MgCl in Tris-maleate buffer (pH 7.4) at 4°C, the teeth were incubated for alkaline phosphatase in a medium consisting of 6 ml 3% sodiumβ-glycerophosphate, 4 ml 0.2M Tris-HCl buffer (pH 9.2), 3 ml 1.6% MgSO4, 12 ml 0.5% lead citrate (pH⋍12), and 2.1 g sucrose. The pH was adjusted to 9.2 with 0.2M HCl, the volume made up to 30 ml, and the solution centrifuged for 10 min at 5000 rpm. Control teeth were incubated in medium minus the substrate. Finally, the specimens were routinely post-fixed and embedded for sectioning and examination with a Philips 300 electron microscope. A gradient of alkaline phosphatase activity was mapped along the developing teeth in the cells of the stratum intermedium, the proximal borders of the ameloblasts, the early dentine matrix, the predentine-dentine border, matrix vesicles, and the plasma membranes of odontoblasts and subodontoblast cells. The gradient of alkaline phosphatase activity was evident in the forming tooth from the cervical loop to the crown apex and was related to the cellular events, matrix synthesis, and matrix mineralization occurring during odontogenesis.

Persistent bronchiolar remodeling following brief ventilation of the very immature ovine lung

Children and adults who were mechanically ventilated following preterm birth are at increased risk of reduced lung function, suggesting small airway dysfunction. We hypothesized that short periods of mechanical ventilation of very immature lungs can induce persistent bronchiolar remodeling that may adversely affect later lung function. Our objectives were to characterize the effects of brief, positive-pressure ventilation per se on the small airways in very immature, surfactant-deficient lungs and to determine whether the effects persist after the cessation of ventilation. Fetal sheep (0.75 of term) were mechanically ventilated in utero with room air (peak inspiratory pressure 40 cmH2O, positive end-expiratory pressure 4 cmH2O, 65 breaths/min) for 6 or 12 h, after which tissues were collected; another group was studied 7 days after 12-h ventilation. Age-matched unventilated fetuses were controls. The mean basement membrane perimeter of airways analyzed was 548.6+/-8.5 microm and was not different between groups. Immediately after ventilation, 21% of airways had epithelial injury; in airways with intact epithelium, there was more airway smooth muscle (ASM) and less collagen, and the epithelium contained more mucin-containing and apoptotic cells and fewer proliferating cells. Seven days after ventilation, epithelial injury was absent but the epithelium was thicker, with greater cell turnover; there were increased amounts of bronchiolar collagen and ASM and fewer alveolar attachments. The increase in ASM was likely due to cellular hypertrophy rather than hyperplasia. We conclude that brief mechanical ventilation of the very immature lung induces remodeling of the bronchiolar epithelium and walls that lasts for at least 7 days; such changes could contribute to later airway dysfunction.

Airway disease: the use of large animal models for drug discovery.

Large animal models have contributed to our current understanding of respiratory pathophysiology and the effects of pulmonary disease modifying drugs. For drug development, the benefit of using large animals over smaller animal species is primarily due to the greater similarity between humans and equivalent sized animals in terms of gross anatomy, morphometry, structure and physiology of their respiratory systems. Thus, when appropriate lung structure and function are required for correctly assessing the efficacy of novel drugs, large animals can play an important role in the development of these drugs to combat respiratory disease. The most widely used and best characterised large animal for drug development has been the sheep model of asthma. Recently, large animal models for chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) have been reported but thus far have not been used extensively for drug development. Some important limitations of using large animals are the large costs associated with this type of research, as well as the poorer understanding of disease mechanisms in these species relative to rodents. In this review we discuss the extent of correlations between preclinical testing performed in large animal models and the initial indication of clinical efficacy in ongoing clinical trials.

Lung Parenchyma at Maturity Is Influenced by Postnatal Growth but Not by Moderate Preterm Birth in Sheep

Background: We have recently shown that moderate preterm birth, in the absence of respiratory support, altered the structure of lung parenchyma in young lambs, but the long-term effects are unknown. Objectives: To determine whether structural changes persist to maturity, and whether postnatal growth affects lung structure at maturity in sheep. Methods: At approximately 1.2 years after birth, lung parenchyma of sheep born 14 days before term (n = 7) was stereologically compared with that of controls born at term (n = 8, term approx. 146 days). Results: Preterm birth per se had no significant effect on lung volume, alveolar number and size, and thicknesses of the alveolar walls and blood-gas barrier. After combining the preterm and term groups, we examined the effects of postnatal growth rates on lung parenchyma. Slower-growing sheep (SG; n = 7: 4 preterm, 3 term) were compared with faster-growing sheep (FG; n = 8: 3 preterm, 5 term). At approximately 1.2 years, the right lung volume, relative to body weight, was significantly lower in SG than FG sheep (p < 0.05) and alveolar number was significantly lower by approximately 44%. The total alveolar internal surface area of the right lung of SG sheep was 38% smaller than in FG sheep; it was also significantly lower when related to both lung and body weight. Conclusions: Our data suggest that moderate preterm birth does not cause persistent alterations in lung parenchyma. However, slow postnatal growth in low-birth-weight sheep results in smaller lungs with fewer alveoli and a lower alveolar surface area relative to body weight.

Growth-hormone-induced signal transducer and activator of transcription 5 signaling causes gigantism, inflammation, and premature death but protects mice from aggressive liver cancer.

Persistently high levels of growth hormone (GH) can cause liver cancer. GH activates multiple signal‐transduction pathways, among them janus kinase (JAK) 2‐signal transducer and activator of transcription (STAT) 5 (signal transducer and activator of transcription 5). Both hyperactivation and deletion of STAT5 in hepatocytes have been implicated in the development of hepatocellular carcinoma (HCC); nevertheless, the role of STAT5 in the development of HCC as a result of high GH levels remains enigmatic. Thus, we crossed a mouse model of gigantism and inflammatory liver cancer caused by hyperactivated GH signaling (GHtg) to mice with hepatic deletion of STAT5 (STAT5Δhep). Unlike GHtg mice, GHtgSTAT5Δhep animals did not display gigantism. Moreover, the premature mortality, which was associated with chronic inflammation, as well as the pathologic alterations of hepatocytes observed in GHtg mice, were not observed in GHtg animals lacking STAT5. Strikingly, loss of hepatic STAT5 proteins led to enhanced HCC development in GHtg mice. Despite reduced chronic inflammation, GHtgSTAT5Δhep mice displayed earlier and more advanced HCC than GHtg animals. This may be attributed to the combination of increased peripheral lipolysis, hepatic lipid synthesis, loss of hepatoprotective mediators accompanied by aberrant activation of tumor‐promoting c‐JUN and STAT3 signaling cascades, and accumulation of DNA damage secondary to loss of cell‐cycle control. Thus, HCC was never observed in STAT5Δhep mice. Conclusion: As a result of their hepatoprotective functions, STAT5 proteins prevent progressive fatty liver disease and the formation of aggressive HCC in the setting of hyperactivated GH signaling. At the same time, they play a key role in controlling systemic inflammation and regulating organ and body size. (Hepatology 2012)