The efficacy and safety of pinocembrin in a sheep model of bleomycin-induced pulmonary fibrosis.

The primary flavonoid, pinocembrin, is thought to have a variety of medical uses which relate to its reported anti-oxidant, anti-inflammatory, anti-microbial and anti-cancer properties. Some studies have reported that this flavonoid has anti-fibrotic activities. In this study, we investigated whether pinocembrin would impede fibrosis, dampen inflammation and improve lung function in a large animal model of pulmonary fibrosis. Fibrosis was induced in two localized lung segments in each of the 10 sheep participating in the study. This was achieved via two infusions of bleomycin delivered bronchoscopically at a two-week interval. Another lung segment in the same sheep was left untreated, and was used as a healthy control. The animals were kept for a little over 5 weeks after the final infusion of bleomycin. Pinocembrin, isolated from Eucalyptus leaves, was administered to one of the two bleomycin damaged lung segments at a dose of 7 mg. This dose was given once-weekly over 4-weeks, starting one week after the final bleomycin infusion. Lung compliance (as a measure of stiffness) was significantly improved after four weekly administrations of pinocembrin to bleomycin-damaged lung segments. There were significantly lower numbers of neutrophils and inflammatory cells in the bronchoalveolar lavage of bleomycin-infused lung segments that were treated with pinocembrin. Compared to bleomycin damaged lung segments without drug treatment, pinocembrin administration was associated with significantly lower numbers of immuno-positive CD8+ and CD4+ T cells in the lung parenchyma. Histopathology scoring data showed that pinocembrin treatment was associated with significant improvement in inflammation and overall pathology scores. Hydroxy proline analysis showed that the administration of pinocembrin did not reduce the increased collagen content that was induced by bleomycin in this model. Analyses of Masson's Trichrome stained sections showed that pinocembrin treatment significantly reduced the connective tissue content in lung segments exposed to bleomycin when compared to bleomycin-infused lungs that did not receive pinocembrin. The striking anti-inflammatory and modest anti-fibrotic remodelling effects of pinocembrin administration were likely linked to the compound's ability to improve lung pathology and functional compliance in this animal model of pulmonary fibrosis.

Recognition of Schistosoma mansoni egg-expressed ovalbumin by T cell receptor transgenic mice.

Schistosoma mansoni eggs can influence immune responses directed at them, and the mechanisms by which this is achieved are being unravelled. Going towards, developing effective tools for the study of how S. mansoni influences naïve T cells, we have developed S. mansoni eggs expressing chicken ovalbumin (OVA), using a lentiviral transduction system. Indeed, such a parasite could be used in conjunction with cells from OT-II transgenic mice as a source of naïve, antigen-specific T cells. The expression of the transgenic protein was confirmed by real-time RT-PCR of OVA-specific mRNA and western blotting using polyclonal antibodies specific for OVA. T cells from OT-II transgenic mice expressing a T cell receptor specific for the OVA323-339 peptide recognised the OVA-transduced S. mansoni eggs. Using flow cytometry on CFSE-labelled OT-II splenocytes, we demonstrated that OVA-transduced eggs elicit higher OT-II proliferative responses than untransduced eggs. The OT-II T cells also produced TNF-α and IFN-γ following exposure to OVA-transduced eggs. In addition, moderate amounts of IL-6 and IL-17A were also detected. In contrast, no IL-10, IL-4 and IL-2 were detected in cultures, whether the cells were stimulated with transduced or untransduced eggs. Thus, the cytokine signatures showed the transfected eggs induced predominantly a Th1 response, with a small amount of IL-6 and IL-17.

Mucosal-Associated Invariant T Cells Augment Immunopathology and Gastritis in Chronic Helicobacter pylori Infection.

Mucosal-associated invariant T (MAIT) cells produce inflammatory cytokines and cytotoxic granzymes in response to by-products of microbial riboflavin synthesis. Although MAIT cells are protective against some pathogens, we reasoned that they might contribute to pathology in chronic bacterial infection. We observed MAIT cells in proximity to Helicobacter pylori bacteria in human gastric tissue, and so, using MR1-tetramers, we examined whether MAIT cells contribute to chronic gastritis in a mouse H. pylori SS1 infection model. Following infection, MAIT cells accumulated to high numbers in the gastric mucosa of wild-type C57BL/6 mice, and this was even more pronounced in MAIT TCR transgenic mice or in C57BL/6 mice where MAIT cells were preprimed by Ag exposure or prior infection. Gastric MAIT cells possessed an effector memory Tc1/Tc17 phenotype, and were associated with accelerated gastritis characterized by augmented recruitment of neutrophils, macrophages, dendritic cells, eosinophils, and non-MAIT T cells and by marked gastric atrophy. Similarly treated MR1-/- mice, which lack MAIT cells, showed significantly less gastric pathology. Thus, we demonstrate the pathogenic potential of MAIT cells in Helicobacter-associated immunopathology, with implications for other chronic bacterial infections.

Omega-1 knockdown in Schistosoma mansoni eggs by lentivirus transduction reduces granuloma size in vivo.

Schistosomiasis, one of the most important neglected tropical diseases worldwide, is caused by flatworms (blood flukes or schistosomes) that live in the bloodstream of humans. The hepatointestinal form of this debilitating disease results from a chronic infection with Schistosoma mansoni or Schistosoma japonicum. No vaccine is available to prevent schistosomiasis, and treatment relies predominantly on the use of a single drug, praziquantel. In spite of considerable research effort over the years, very little is known about the complex in vivo events that lead to granuloma formation and other pathological changes during infection. Here we use, for the first time, a lentivirus-based transduction system to deliver microRNA-adapted short hairpin RNAs (shRNAmirs) into the parasite to silence and explore selected protein-encoding genes of S. mansoni implicated in the disease process. This gene-silencing system has potential to be used for functional genomic-phenomic studies of a range of socioeconomically important pathogens.

Biodegradable and biocompatible poly(ethylene glycol)-based hydrogel films for the regeneration of corneal endothelium.

Corneal endothelial cells (CECs) are responsible for maintaining the transparency of the human cornea. Loss of CECs results in blindness, requiring corneal transplantation. In this study, fabrication of biocompatible and biodegradable poly(ethylene glycol) (PEG)-based hydrogel films (PHFs) for the regeneration and transplantation of CECs is described. The 50-µm thin hydrogel films have similar or greater tensile strengths to human corneal tissue. Light transmission studies reveal that the films are >98% optically transparent, while in vitro degradation studies demonstrate their biodegradation characteristics. Cell culture studies demonstrate the regeneration of sheep corneal endothelium on the PHFs. Although sheep CECs do not regenerate in vivo, these cells proliferate on the films with natural morphology and become 100% confluent within 7 d. Implantation of the PHFs into live sheep corneas demonstrates the robustness of the films for surgical purposes. Regular slit lamp examinations and histology of the cornea after 28 d following surgery reveal minimal inflammatory responses and no toxicity, indicating that the films are benign. The results of this study suggest that PHFs are excellent candidates as platforms for the regeneration and transplantation of CECs as a result of their favorable biocompatibility, degradability, mechanical, and optical properties.

Biological activity of ovine IL-23 expressed using a foot-and-mouth disease virus 2A self-cleaving peptide.

Hetero-dimeric cytokines often require equi-molar expression of both subunits to achieve biological activity. Previously, we expressed ovine IL-12 p40 and p35 linked using a self-cleaving 2A peptide from foot-and-mouth disease virus (FMDV). We now generated a new improved vector for the expression of hetero-dimeric cytokines and demonstrate the more general applicability of this strategy by cloning and expressing ovine IL-23 using the 2A peptide to link IL-12/IL-23 p40 and p19. The resulting protein was shown to be biologically active when expressed in mammalian COS cells. IL-23 plays a significant role in the differentiation of Th17 cells as well as autoimmunity and the regulation of inflammatory processes. As such this reagents will be invaluable in the unravelling of regulation of the ovine immune system for both veterinary and human animal model applications.

Inflammatory cytokines IL-6 and TNF-α regulate lymphocyte trafficking through the local lymph node.

Lymphocyte trafficking from blood to lymph and back is a tightly regulated process. Given appropriate stimuli, trafficking of cells through the lymph node changes from a 'steady-state' to a bimodal flow. Initially, a 'shutdown' phase occurs, leading to a dramatic reduction in efferent cell output. This is followed by a 'recruitment' phase whereby the efferent cell output becomes greatly elevated before returning to baseline levels. The shutdown/recruitment process is hypothesised to promote encounters between Ag-specific lymphocytes and APCs in an environment conducive to immune response induction. Cytokines, such as TNF-α have been shown to play an important role in regulating lymphocyte trafficking. Here, we unravel the role of cytokines in the regulation of cell trafficking using an in vivo sheep lymphatic cannulation model whereby the prefemoral lymph nodes were cannulated and recombinant cytokines were injected subcutaneously into the draining area of the cannulated node. We demonstrate that local injection of purified IL-6 or TNF-α stimulates shutdown/recruitment in the draining lymph node. While the effect of IL-6 appears to be direct, TNF-α may mediate shutdown/recruitment through IL-6.

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.

Thoracic duct cannulation without thoracotomy in sheep: a method for accessing efferent lymph from the lung.

We have developed and validated a novel method to access efferent lymph draining the lung and gut of sheep. In this model, efferent lymph derived from the lung could be collected via cannulation of the thoracic duct just prior the thoracic duct-jugular vein junction. The thoracic duct was accessed in the neck region without needing to broach the thoracic cavity, thus avoiding extensive tissue damage to the animal and need for ventilation during surgery. In addition, this surgical approach allows for a second cannulation of an adjacent lymphatic draining the head/neck region, providing for an 'in-built' internal control with which to compare lymph parameters. To test the verity of cannulation procedure, a test protein ovalbumin (OVA) was infused into the left and right lungs via bronchoscopy. We found that OVA was recovered almost exclusively in the lymph draining the lungs compared to the lymph draining the head/neck where it was essentially non-existent. The method described here will be invaluable for optimizing intra-lung delivery of drugs or vaccines. In addition, access to lymph will also allow for analysis of immune responses to infections originating at this site.

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.

Co-delivery of plasmid-encoded cytokines modulates the immune response to a DNA vaccine delivered by in vivo electroporation.

In this study, in vivo electroporation of a DNA vaccine adjuvanted with plasmids encoding different cytokines was investigated in large animals. Sheep were injected intramuscularly with a DNA vaccine encoding an antigen of Haemonchus contortus (pNPA) and plasmids encoding different cytokines followed by in vivo electroporation. Plasmids (pCI) carrying the genes of different cytokines including ovine IL-4(pCI-IL4), IL-10(pCI-IL10), GM-CSF(pCI-GMCSF), and MCP-1alpha(pCI-MCP1alpha), and pCI-IL4+pCI-GMCSF were co-delivered with pNPA. The results showed that co-delivery of pCI-GMCSF or pCI-IL4+pCI-GMCSF significantly enhanced both antibody responses and T cell proliferation responses to the antigen after two DNA immunisations compared to co-delivery of pCI. In contrast, antibody responses of the sheep that received pCI-IL10 were decreased significantly. Other cytokine expressing plasmids did not significantly alter the measured immune responses. Furthermore, co-delivery of pCI-GMCSF increased IgG2 response more than IgG1 responses, suggesting a Th1 bias. However, the increase in IgG2 over IgG1 was less apparent when co-delivery of pCI-IL4 with pCI-GMCSF. Interestingly, the co-delivery of pCI-IL4 alone did not increase the IgG1 titre, suggesting that both pCI-GMCSF and pCI-IL4 are required for optimal IgG1 production. Thus, co-delivery of plasmid-encoded cytokine genes with in vivo electroporation has the ability to effectively modulate immune responses to a DNA vaccine in a large animal.

Activin A: from sometime reproductive factor to genuine cytokine.

The growth factor, activin A, was initially characterized as a putative reproductive hormone but is now known to have many other divergent roles. One of these is during inflammation. Following intravenous injection of bacterial lipopolysaccharide (LPS) into sheep, activin A is released extremely rapidly into the circulation. The release of activin A appears to be independent of fever, prostaglandins or other key proinflammatory cytokines such as TNF-alpha or IL-1beta. While the precise roles and function of this factor in inflammation are yet to be elucidated, the activin response occurs in other mammalian species besides the sheep and elevated activin has been documented for a number of clinical inflammatory conditions. Activin A therefore seems to be part of the regulatory component of the innate immune response.

Prolongation of sheep corneal allograft survival by transfer of the gene encoding ovine IL-12-p40 but not IL-4 to donor corneal endothelium.

Immunological rejection is the major cause of human corneal allograft failure. We hypothesized that local production of IL-4 or the p40 subunit of IL-12 (p40 IL-12) by the grafted cornea might prolong allograft survival. Replication-deficient adenoviral vectors encoding ovine IL-4 or p40 IL-12 and GFP were generated and used to infect ovine corneas ex vivo. mRNA for each cytokine was detected in infected corneas, and the presence of secreted protein in corneal supernatants was confirmed by bioassay (for IL-4) or immunoprecipitation (for p40 IL-12). Sheep received uninfected or gene-modified orthotopic corneal allografts. Postoperatively, untreated corneas (n = 13) and corneas expressing GFP (n = 6) were rejected at a median of 21 and 20 days, respectively. Corneas expressing IL-4 (n = 6) underwent rejection at 18.5 days (p > 0.05 compared with controls) and histology demonstrated the presence of eosinophils. In contrast, corneas expressing p40 IL-12 (n = 9) showed prolonged allograft survival (median day to rejection = 45 days, p = 0.003). Local intraocular production of p40 IL-12 thus prolonged corneal graft survival significantly, but local production of the prototypic immunomodulatory cytokine IL-4 induced eosinophilia, inflammation, and rejection. These findings have important implications for the development of novel strategies to improve human corneal graft survival.

Gene gun immunization in a preclinical model is enhanced by B7 targeting.

DNA vaccines have great potential but despite the promise shown in rodent models, responses in large animals, including humans, have been disappointing. Furthermore, gene gun delivery of DNA has been used to improve these responses. However, most cells that are transfected are not the professional antigen presenting cells (APC) which are critical for generating the primary immune response. Here, we show that in the large animal model of the pig, the combination of the use of gene gun delivery and a DNA vector that targets antigen presenting cells by expressing a CTLA4-ovalbumin (OVA) fusion antigen, leads to enhanced ovalbumin specific serum IgG, IgA, IgG1 and IgG2 immune responses.

White matter injury after repeated endotoxin exposure in the preterm ovine fetus.

Intrauterine infection has been linked to neurologic injury in preterm infants. However, a reproducible model of white matter injury in the preterm fetus in a long gestation species that can be monitored in utero is currently unavailable. Thus, our objective was to determine the effects of bacterial endotoxin (lipopolysaccharide, LPS) on physiologic and inflammatory responses and brain structure in the preterm ovine fetus. At 0.7 of gestation, six catheterized fetuses received three to five intravenous injections of LPS (1 micro g/kg) over 5 d; seven fetuses served as controls. Fetal responses were monitored and brain tissue examined 10-11 d after the initial LPS injection. After LPS on d 1 and 2, fetuses became transiently hypoxemic and hypotensive and blood IL-6 levels were increased, but these responses were smaller or absent after subsequent LPS exposures. Neural injury was observed in all LPS-exposed fetuses, most prominently in the cerebral white matter. Injury ranged from diffuse subcortical damage to periventricular leukomalacia, and in the brainstem the cross-sectional area of the corticospinal tract was reduced by 30%. Thus, repeated exposure of the preterm ovine fetus to LPS causes neuropathology resembling that of cerebral palsy and provides a robust model for exploring the etiology, prevention, and treatment of white matter damage.