Measuring the Manipulation of T Helper Immune Responses by Schistosoma mansoni.

Schistosoma mansoni uses different mechanisms to escape its host's immunity. Understanding the ability of memory T cells to withstand this pathogen's manipulation is important for the development of effective vaccines against this immunomodulatory pathogen. In this study, ovalbumin (OVA) transgenic S. mansoni is used as a tool to investigate whether fully differentiated Th1, Th2 and Th17 cells are able to withstand pathogen manipulation. Naïve T cells from OT-II T cell receptor transgenic mice with a specificity for OVA were differentiated into Th1, Th2, and Th17 polarised memory cells in vitro. These cells were adoptively transferred into recipient mice to investigate whether these polarised immune memory T cells are resilient in the face of pathogen-mediated manipulation. After transferring memory cells, mice were challenged with OVA-transduced S. mansoni eggs as well as wild-type controls. The in vitro differentiated Th1, Th2 and Th17 memory cells continued to produce the same cytokines when challenged by OVA-expressing S. mansoni eggs as to these they produced when transferred in vivo, suggesting that the Th phenotypes of the memory T cells remains unaltered in the face of stimulation by S. mansoni. The ability of memory T cells to remain resilient to manipulation by the parasite suggests that vaccines might be able to produce immune memory responses able to withstand S. mansoni immune manipulation and hence protect the host from infection.

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.

Investigating immune responses to parasites using transgenesis.

Parasites comprise diverse and complex organisms, which substantially impact human and animal health. Most parasites have complex life-cycles, and by virtue of co-evolution have developed multifaceted, often life-cycle stage-specific relationships with the immune system of their hosts. The complexity in the biology of many parasites often limits our knowledge of parasite-specific immune responses, to in vitro studies only. The relatively recent development of methods to stably manipulate the genetic make-up of many parasites has allowed a better understanding of host-parasite interactions, particularly in vivo. In this regard, the use of transgenic parasites can facilitate the study of immunomodulatory mechanisms under in vivo conditions. Therefore, in this review, we specifically highlighted the current developments in the use of transgenic parasites to unravel the host's immune response to different life-cycle stages of some key parasite species such as Leishmania, Schistosoma, Toxoplasma, Plasmodium and Trypanosome and to some degree, the use of transgenic nematode parasites is also briefly discussed.

Identification and characterization of an M cell marker in nasopharynx- and oropharynx-associated lymphoid tissue of sheep.

M cells play a pivotal role in the induction of immune responses within the mucosa-associated lymphoid tissues. M cells exist principally in the follicle-associated epithelium (FAE) of the isolated solitary lymphoid follicles as well as in the lymphoid follicles of nasopharynx-associated lymphoid tissue and gut associated lymphoid tissue (GALT). Through lymphatic cannulation it is possible to investigate local immune responses induced following nasal Ag delivery in sheep. Hence, identifying sheep M cell markers would allow the targeting of M cells to offset the problem of trans-epithelial Ag delivery associated with inducing mucosal immunity. Sheep cDNA from the tonsils of the oropharynx and nasopharynx was PCR amplified using Glycoprotein-2 (GP2)-specific primers and expressed as a poly-His-tagged recombinant sheep GP2 (56 kDa) in HEK293 cells. The recombinant GP2 protein was purified using Ni-NTA affinity chromatography and polyclonal serum against the protein was raised in rats. The antiserum recognized the recombinant sheep GP2 and purified rat IgG against GP2 stained M cells in sections of sheep tonsils from nasopharynx and oropharynx. M cells were found to be present in epithelium of the palatine tonsils (oropharynx), pharyngeal tonsils as well as tubal tonsils (nasopharynx). They were also present in the FAE of the scattered lymphoid follicles over the base of the nasopharynx. Thus, GP2 has been identified to be an important M cell marker of nasopharynx and oropharynx-associated lymphoid tissues in sheep.

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.

Knocking down schistosomes - promise for lentiviral transduction in parasites.

Underpinned by major advances in our understanding of the genomes of schistosomes, progress in the development of functional genomic tools is providing unique prospects to gain insights into the intricacies of the biology of these blood flukes, their host relationships, and the diseases that they cause. This article reviews some key applications of double-stranded RNA interference (RNAi) in Schistosoma mansoni, appraises delivery systems for transgenesis and stable gene silencing, considers ways of increasing efficiency and specificity of gene silencing, and discusses the prospects of using a lentivirus delivery system for future functional genomic-phenomic explorations of schistosomes and other parasites. The ability to achieve effective and stable gene perturbation in parasites has major biological implications and could facilitate the development of new interventions.

Prospects for Vector-Based Gene Silencing to Explore Immunobiological Features of Schistosoma mansoni.

Schistosomiasis is a prevalent, socioeconomically important disease of humans caused by parasites of the genus Schistosoma (schistosomes or blood flukes). Currently, more than 200 million people worldwide are infected with schistosomes. Despite major research efforts, there is only one drug routinely used for effective treatment, and no vaccine is available to combat schistosomiasis. The purpose of the present article is to (1) provide a background on the parasites and different forms of disease; (2) describe key immunomolecular aspects of disease induced in the host; and (3) critically appraise functional genomic methods employed to explore parasite biology, parasite-host interactions and disease at the molecular level. Importantly, the article also describes the features and advantages of lentiviral delivery of artificial microRNAs to silence genes. It also discusses the first successful application of such an approach in schistosomes, in order to explore the immunobiological role of selected target proteins known to be involved in egg-induced disease. The lentiviral transduction system provides exciting prospects for future, fundamental investigations of schistosomes, and is likely to have broad applicability to other eukaryotic pathogens and infectious diseases. The ability to achieve effective and stable gene perturbation in parasites has major biotechnological implications, and might facilitate the development of radically new methods for the treatment and control of parasitic diseases.

Transcriptional analysis identifies key genes involved in metabolism, fibrosis/tissue repair and the immune response against Fasciola hepatica in sheep liver.

BACKGROUND: Although fascioliasis has been relatively well studied, little is known about the molecular basis of this disease. This is particularly relevant, considering the very different response that sheep have to Fasciola hepatica relative to cattle. The acute phase of this disease is severe in sheep, whereas chronic fascioliasis is more common in cattle. METHODS: To begin to explore the host-response to Fasciola in sheep and improve the understanding of the host-pathogen interactions during the parasite's migration through liver parenchyma to the bile duct, we used RNA sequencing (RNA-seq) to investigate livers from sheep infected for eight weeks compared with those from uninfected controls. RESULTS: This study identified 572 and 42 genes that were up- and down-regulated, respectively, in infected livers relative to uninfected controls. Our molecular findings provide significant new insights into the mechanisms linked to metabolism, fibrosis and tissue-repair in sheep, and highlight the relative importance of specific components of immune response pathways, which appear to be driven toward a suppression of inflammation. CONCLUSIONS: This study is, to our knowledge, the first detailed investigation of the transcriptomic responses in the liver tissue of any host to F. hepatica infection. It defines the involvement of specific genes associated with the host's metabolism, immune response and tissue repair/regeneration, and highlights an apparent overlapping function of many genes involved in these processes.

ISCOMATRIX™ adjuvant reduces mucosal tolerance for effective pulmonary vaccination against influenza.

While most pathogens infect via mucosal surfaces, most current vaccines are delivered by injection. This situation remains despite awareness of the potential benefits of mucosal delivery for inducing protection against mucosa-infecting pathogens. A major obstacle to the development of such vaccines is the paucity of safe and effective adjuvants that induce mucosal responses in non-rodents. Previously we demonstrated in sheep the potency of pulmonary-delivered influenza ISCOMATRIX™ vaccine, which induces both mucosal and systemic immunity, even with low antigen doses. In the current study, lung pre-exposure to influenza antigen alone significantly reduced the immune response to subsequent pulmonary-delivered influenza ISCOMATRIX™ vaccine. A single dose of influenza antigen, delivered to the lung without exogenous adjuvant, upregulated IL-10 expression in bronchoalveolar lavage cells and FOXP3 expression in lung tissue, suggestive of induction of a regulatory T cell (Treg) response. However, this effect was inhibited by addition of ISCOMATRIX™ adjuvant. Moreover, effective pulmonary immunization with influenza ISCOMATRIX™ vaccine was associated with a depletion of Treg markers within lung tissues. Lung exposure to influenza antigen induced a localized mucosal tolerance that reduced the efficacy of subsequent influenza ISCOMATRIX™ vaccination. An important role of ISCOMATRIX™ adjuvant in pulmonary vaccination appears to be the depletion of Treg in lung tissues. Pulmonary vaccination remains capable of inducing a strong immune response against mucosal pathogens, but likely requires an adjuvant to overcome mucosal tolerance. ISCOMATRIX™ appears to have considerable potential as a mucosal adjuvant for use in humans, a major unmet need in mucosal vaccine development.

Exploring local immune responses to vaccines using efferent lymphatic cannulation.

The early stages of the induction of a primary immune response to a vaccine can shape the overall quality of the immune memory generated and hence affect the success of the vaccine. This early interaction between a vaccine and the immune system occurs first at the site of vaccination and can be explored using afferent cannulation. Subsequently, the vaccine and adjuvant activates the local draining lymph node. These interactions can be studied in real time in vivo using efferent lymphatic duct cannulation in large animal models and are the subject of this review. Depending on how the vaccine is delivered, the draining lymph nodes of different organs can be accessed, facilitating the testing of tissue-specific vaccinations. The efferent lymphatic cannulation model provides an avenue to study the effect of both adjuvants and antigen on the local immune system, and hence opens a pathway toward developing more effective ways of inducing immunity.

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.

Techniques for the diagnosis of Fasciola infections in animals: room for improvement.

The common liver fluke, Fasciola hepatica, causes fascioliasis, a significant disease in mammals, including livestock, wildlife and humans, with a major socioeconomic impact worldwide. In spite of its impact, and some advances towards the development of vaccines and new therapeutic agents, limited attention has been paid to the need for practical and reliable methods for the diagnosis of infection or disease. Accurate diagnosis is central to effective control, particularly given an emerging problem with drug resistance in F. hepatica. Traditional coprological techniques have been widely used, but are often unreliable. Although there have been some advances in establishing immunologic techniques, these tools can suffer from a lack of diagnostic specificity and/or sensitivity. Nonetheless, antigen detection tests seem to have considerable potential, but have not yet been adequately evaluated in the field. Moreover, advanced nucleic acid-based methods appear to offer the most promise for the diagnosis of current infection. This chapter (i) provides a brief account of the biology and significance of F. hepatica/fascioliasis, (ii) describes key techniques currently in use, (iii) compares their advantages/disadvantages and (iv) reviews polymerase chain reaction-based methods for specific diagnosis and/or the genetic characterization of Fasciola species.

Genome and transcriptome of the porcine whipworm Trichuris suis.

Trichuris (whipworm) infects 1 billion people worldwide and causes a disease (trichuriasis) that results in major socioeconomic losses in both humans and pigs. Trichuriasis relates to an inflammation of the large intestine manifested in bloody diarrhea, and chronic disease can cause malnourishment and stunting in children. Paradoxically, Trichuris of pigs has shown substantial promise as a treatment for human autoimmune disorders, including inflammatory bowel disease (IBD) and multiple sclerosis. Here we report whole-genome sequencing at ∼140-fold coverage of adult male and female T. suis and ∼80-Mb draft assemblies. We explore stage-, sex- and tissue-specific transcription of mRNAs and small noncoding RNAs.

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.

Characterisation of local immune responses induced by a novel nano-particle based carrier-adjuvant in sheep.

Most adjuvants require danger signals to promote immune responses against vaccine antigens. Our previous studies have characterised a powerful nano-particulate antigen delivery system, which by itself does not induce inflammation, and which further appears to induce substantial immune responses in mice and sheep without the requirement for added stimulators of toll like receptors or other pathogen recognition receptors. In the present study we dissect the nature of the early induction phase of the immune response stimulated by such a vaccine comprising 40 nm polystyrene nano-particles conjugated to the antigen. We analyse the kinetics of export from an individual draining lymph node from the sheep, of antibodies and cytokines as well as antigen responsive CD4 and CD8 T cells. Our results indicate that simple inert nano-bead based antigen delivery into the draining area of the lymph node is highly efficient at priming combined humoral and T cell antigen specific immunity without the need for added 'danger signals'. Furthermore this nano-bead adjuvant is a potent agent capable of promoting cross-priming for CD8 T cell induction in sheep. Interestingly, using nano-beads, similarly to what has been observed with natural pathogen based lymph node stimulation, a phase of CD4 T cell priming and export preceded CD8 T cell induction, suggesting the engagement of natural priming processes and kinetics.

Phenotypic analysis of ovine antigen presenting cells loaded with nanoparticles migrating from the site of vaccination.

Virus-sized particulate adjuvants such as ISCOMs, polystyrene nanoparticles and virus-like particles have been shown to target dendritic cells, resulting in the activation of T and B cells in vivo. Using an ovine pseudo-afferent lymph cannulation model to capture APC that traffic from the site of injection to the local lymph node, we show that 40-50 nm nanoparticles are taken up at the site of injection by dendritic cells (DCs) migrating to the draining lymph node. These DCs can express CD11c, CD1b, CD5, MHC class II and CD8. Nanoparticles transported by DCs migrating from the site of injection to the local lymph node therefore needs to be considered as a new mechanism underlying the immunogenicity of virus-sized vaccine delivery systems.

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.

Mucosal vaccination: lung versus nose.

The induction of potent mucosal immune responses able to prevent the establishment of infection at the onset of mucosal pathogen colonisation represents a desirable but challenging goal for vaccine development. Here we compare nasal vaccine delivery with intra-pulmonary vaccination using a sheep lymphatic cannulation model. Our results demonstrate that nasal delivery of a non-infective ISCOMATRIX(®) influenza vaccine does not induce primary immune responses in the lymph draining the nasal lymph nodes, suggesting that local immune responses in the lymph nodes draining the nasal cavity are relatively weak. However, this mode of delivery can boost existing immunity in the nasal lymph. Using the same adjuvant we were able to induce very potent immune responses in both blood and bronchoalveolar lavage (BAL), following intra-pulmonary delivery of ISCOMATRIX(®) influenza vaccine, even when very small doses of antigen were employed. Lung delivery could also induce comparable immune responses against other recombinant antigens mixed with ISCOMATRIX(®) adjuvant and could therefore become a method of choice for the induction of immunity to mucosal pathogens infecting the lower respiratory tract.

Long-term antibody and immune memory response induced by pulmonary delivery of the influenza Iscomatrix vaccine.

Pulmonary delivery of an influenza Iscomatrix adjuvant vaccine induces a strong systemic and mucosal antibody response. Since an influenza vaccine needs to induce immunological memory that lasts at least 1 year for utility in humans, we examined the longevity of the immune response induced by such a pulmonary vaccination, with and without antigen challenge. Sheep were vaccinated in the deep lung with an influenza Iscomatrix vaccine, and serum and lung antibody levels were quantified for up to 1 year. The immune memory response to these vaccinations was determined following antigen challenge via lung delivery of influenza antigen at 6 months and 1 year postvaccination. Pulmonary vaccination of sheep with the influenza Iscomatrix vaccine induced antigen-specific antibodies in both sera and lungs that were detectable until 6 months postimmunization. Importantly, a memory recall response following antigenic challenge was detected at 12 months post-lung vaccination, including the induction of functional antibodies with hemagglutination inhibition activity. Pulmonary delivery of an influenza Iscomatrix vaccine induces a long-lived influenza virus-specific antibody and memory response of suitable length for annual vaccination against influenza.