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 microg influenza) induced serum antibody levels equivalent to those resulting from a current human vaccine equivalent (15 microg 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.

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. OBJECTIVE: The aim of the present study was to develop a model for airway remodelling in sheep after repeated challenge with a relevant human allergen to assess the relationship of airway remodelling with inflammation. METHODS: Repeated challenges of house dust mite (HDM) extract or saline (control) were administered to local lung segments of sheep for a period of 6 months. After the last challenge, lung tissues from both challenged and unchallenged lung compartments of the same sheep were compared using morphometric image analysis and (immuno) histological studies. RESULTS: All HDM-challenged sheep developed significant bronchoalveolar lavage eosinophilia during challenge. At the end of the challenge period, significant increases in airway collagen and airway smooth muscle content were found in a proportion (3/7) of the HDM-challenged sheep. Hyperplasia of goblet cells and epithelial cells were observed in small bronchi and bronchioles exposed to allergen. Irrespective of airway remodelling changes, all HDM-challenged, but no saline-challenged sheep, displayed significant increases in mast cells in alveolar septa and airway walls of challenged lungs compared with untreated lung compartments of the same sheep. Significant increases were also observed in CD5 and gamma delta T cell subpopulations in all allergen-exposed lung parenchyma. CONCLUSION: A proportion of atopic sheep develop typical airway remodelling changes after chronic allergen challenge, which is not directly related to the level of allergic inflammation.

Hepatocellular kinetics and the expression of growth hormone (GH) in the livers and liver tumours of GH-transgenic mice.

Although it is well known that transgenic mice that overexpress growth hormone (GH) frequently develop liver tumours, the precise nature of the relationship between the overexpression of GH and hepatocarcinogenesis is not clear. The current study was designed to investigate the relationship between the expression of the GH transgene and changes in hepatocyte morphology and kinetics, prior to and during hepatocarcinogenesis in GH-transgenic mice. In young mice (1-month-old) prior to tumour development, GH protein, as detected by immunohistochemistry, was observed in the cytoplasm of essentially all hepatocytes. In liver tissues of older animals, apoptotic cells and hypertrophic hepatocytes did not express immunoreactive GH, even though GH was expressed strongly in the smaller hepatocytes. A relatively high proportion of large dysplastic hepatocytes (>50 microm) were apoptotic (TUNEL positive), whereas smaller hepatocytes featured more prominently in the proliferative phase, as measured by BrdU incorporation. GH expression in tumour tissue, as detected by immunohistochemistry, was often variable and generally decreased with tumour development. Northern blot analysis showed that equivalent levels of GH mRNA were present in tumour tissue and adjacent liver. However, there was no clear trend when the levels of GH mRNA extracted from adenoma, and hepatocellular carcinoma, were compared. These observations help clarify some of the mechanisms by which GH promotes hepatocarcinogenesis in GH-transgenic mice.

Overexpressed growth hormone (GH) synergistically promotes carcinogen-initiated liver tumour growth by promoting cellular proliferation in emerging hepatocellular neoplasms in female and male GH-transgenic mice.

BACKGROUND/AIMS: Growth hormone (GH), when overexpressed in male and female GH-transgenic mice, is known to induce liver tumours within 1 year. This study aimed to gain a clearer understanding of the interaction between GH and tumour cells in vivo. METHODS/RESULTS: The carcinogen diethylnitrosomine (DEN) was administered to neo-natal transgenic and non-transgenic mice maintained in a "hepatocarcinogenesis resistant" genetic background (C57BL/6J). Macroscopic, microscopic and liver weight/body weight ratio analyses revealed that carcinogen-induced hepatocarcinogenesis was dramatically accelerated in young GH-transgenic mice compared to non-transgenic counterparts. Image analysis of microscopic hepatocellular neoplasms showed rapidly increasing tumour burdens, and neoplastic foci size over time in young adult GH-transgenic mice. The magnitude of enhanced tumour growth was equivalent in both male and female transgenic mice, whereas much lower and sexually dimorphic tumour growth rates (males>females) were observed in non-transgenic mice treated with DEN. BrdU labelling experiments demonstrated that rapid tumour growth in carcinogen-treated GH-transgenic mice was due to the promotion of cell proliferation in emerging lesions. Tumour cell proliferation in young GH-transgenic mice was 2.6- and 4-fold higher, respectively, than that observed in similar age male and female non-transgenic mice. Interestingly, both GH-transgenic and non-transgenic mice displayed progressively slower tumour growth rates in older animals. CONCLUSION: Overall, GH synergistically promotes carcinogen-induced hepatocarcinogenesis in both sexes of GH-transgenic mice by stimulating tumour cell proliferation.

High, persistent hepatocellular proliferation and apoptosis precede hepatocarcinogenesis in growth hormone transgenic mice.

AIMS/BACKGROUND: Growth hormone (GH) transgenic mice are known to develop hepatocellular adenomas and carcinomas. In order to understand more about hepatocarcinogenesis in the GH-transgenic mouse model we quantitated the rates of hepatocellular proliferation and apoptosis in these mice. METHODS: Two lines of GH-transgenic mice and non-transgenic control mice were generated and sacrificed at regular intervals between one and nine months. Hepatocellular replication was measured by in vivo incorporation of bromodeoxyuridine (BrdU) and counting BrdU-positive nuclei in histological liver sections. Serial sections taken from these mouse livers were also assessed for rates of hepatocellular apoptosis using the in situ end-labelling of fragmented DNA (TUNEL) method. RESULTS: High levels of hepatocellular replication were sustained life-long in this model. Increased rates of hepatocellular proliferation preceded the onset of hepatic inflammation, a prominent feature in the liver pathology of GH-transgenic mice. In tumour tissue, cellular proliferation was up to 17-fold greater than in surrounding non-tumour tissue. Apoptosis rates were also elevated in non-tumour regions of GH-transgenic mouse livers compared to controls. Interestingly, large dysplastic hepatocytes were common in the fraction of cells undergoing apoptosis, especially in older mice with inflamed livers. The increase in the rate of hepatocellular apoptosis in GH-transgenic animals largely balanced the augmented levels of proliferation seen in these mice. In tumour tissue, however, the profound increase in the number of proliferating tumour cells outstripped the increase in apoptosis. CONCLUSION: Relatively high and enduring levels of hepatocellular replication and apoptosis precede hepatocarcinogenesis in GH-transgenic mice. Increased cellular proliferation and resistance to apoptosis were evident in tumour growth in older animals.

Allelic variation of ovine MHC class II DQA1 and DQA2 genes.

In the present study we characterize allelic variation of polymorphic OLA-DQA1 and OLA-DQA2 genes in sheep. To achieve this, PCR primers were designed to independently amplify the second exons of OLA-DQA1 and OLA-DQA2 genes. Single strand conformation polymorphism (SSCP) gel analyses reveals that there are at least 12 distinct OLA-DQA2 sequences, 10 of which have been characterized by sequencing. Six distinct OLA-DQA1 alleles have been sequenced in sheep and we can detect at least seven DQA1 alleles, including a null allele, by SSCP analysis. The second exon of the OLA-DQA2 gene is more polymorphic than the equivalent region of the OLA-DQA1 gene. Thirty-two per cent of nucleotide and 49% of amino acid sites showed variation at the DQA2 locus, compared to 20% of nucleotide and 33% of amino acid sites for DQA1. Phylogenetic analysis of DQA sequences from a number of species show that sheep DQA1 sequences group together and are more similar to bovine DQA1 sequences than to sheep DQA2 alleles. The majority of OLA-DQA2 sequences are on the same main branch of the phylogenetic tree as bovine DQA2 sequences. However, three sheep DQA2 sequences have a tendency to group with putative bovine DQA3 sequences rather than to other ovine DQA2 alleles. A variety of SSCP gel conditions were tried in order to develop a typing system for the OLA-DQA2 gene. We describe a set of PCR and SSCP conditions which distinguish between all known OLA-DQA2 alleles.

Comparison of intrahepatic lymphocytes from normal and growth hormone transgenic mice with chronic hepatitis and liver cancer.

Mice expressing an ovine growth hormone-mouse metallothionein promoter fusion gene (METoGH mice) develop chronic hepatitis which becomes progressively more severe over time, hepatocellular adenomas, and eventually carcinoma in the oldest animals. T-lymphocyte expression of activation/memory-associated markers was compared between liver and blood lymphocytes isolated from METoGH and non-transgenic mice at 7, 10 and 12 months of age. The percentage of intrahepatic lymphocytes (IHL) which were CD4+ was markedly diminished in METoGH mice at all times. CD4+ and CD8+ IHL in METoGH mice expressed Ly-6A/6D at increased density, and were CD45RBlo at later time-points. Ly-6C+ and NK1.1+ CD4+ cells, which are common in normal mouse liver, were found at decreased frequency in METoGH livers. Further analysis demonstrated that, as a proportion of total T-cell receptor (TCR) alpha beta cells, NK1.1+ TCR alpha beta int CD4+ cell numbers (NKT cells) were diminished in the livers of METoGH mice. Observations made in METoGH mice support the hypothesis that sustained liver inflammation and hepatocellular injury may be linked to liver cancer. Additionally, it is possible that the relative lack of NKT cells may create an environment permissive for the growth of liver tumours.

Methylation and expression of a metallothionein promoter ovine growth hormone fusion gene (MToGH1) in transgenic mice.

We have examined transgene methylation in the DNA from the livers of a pedigree of mice carrying three copies of an integrated MToGH1 transgene. Utilizing the methylation-sensitive isoschizomers Msp I and Hpa II, Southern blot analysis revealed that all second generation animals derived from a transgenic female had hypermethylated DNA, whereas first generation animals sired by a transgenic male displayed a range of methylation phenotypes ranging from no methylation to hypermethylation of the transgene sequences. Of the mice that exhibited hypermethylation of the transgene in CpG dinucleotides (CmCGG), a minority of these animals also exhibited apparent CpC methylation (i.e. inhibition of Msp I cutting, presumably blocked by methylation of the outer C of CCGG). Methylation was also examined in the inner C of CC(A/T)GG sequences in the MToGH1 transgene using the isoschizomer pair BstN I and EcoR II. A minority of MToGH1 animals in the F1 generation showed clear evidence of methylation in these sites as well as in the inner and outer Cs of CCGG sites. An examination of MToGH1 expression in terms of oGH levels in serum revealed that there was a high degree of variation in the levels of circulating oGH between animals of this pedigree. There was a weak inverse relationship between the serum level of oGH and the extent of methylation of the transgene. In particular, mice exhibiting CpC together with CpG methylation were found to have very low levels of circulating oGH. Our results highlight the nature and complexity of epigenetic factors associated with transgene sequences which may ultimately influence expression of introduced genes in the mammalian genome.

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

The 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 determined 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 degrees C, the medium consisting of 6 ml 3% sodium beta-glycerophosphate, 4 ml 0.2M Tris-HCl buffer (pH 9.2), 3 ml 1.6% MgSO4, 12 ml 0.5% lead citrate (pH congruent to 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 microscopy. 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.