Human amniotic epithelial cells induce localized cell-mediated immune privilege in vitro:implications for pancreatic islet transplantation

Chronic systemic immunosuppression in cell replacement therapy restricts its clinical application. This study sought to explore the potential of cell-based immune modulation as an alternative to immunosuppressive drug therapy in the context of pancreatic islet transplantation. Human amniotic epithelial cells (AEC) possess innate anti-inflammatory and immunosuppressive properties that were utilized to create localized immune privilege in an in vitro islet cell culture system. Cellular constructs composed of human islets and AEC (islet/AEC) were bioengineered under defined rotational cell culture conditions. Insulin secretory capacity was validated by glucose challenge and immunomodulatory potential characterized using a peripheral blood lymphocyte (PBL) proliferation assay. Results were compared to control constructs composed of islets or AEC cultured alone. Studies employing AEC-conditioned medium examined the role of soluble factors, and fluorescence immunocytochemistry was used to identify putative mediators of the immunosuppressive response in isolated AEC monocultures. Sustained, physiologically appropriate insulin secretion was observed in both islets and islet/AEC constructs. Activation of resting PBL proliferation occurred on exposure to human islets alone but this response was significantly (p <0.05) attenuated by the presence of AEC and AEC-conditioned medium. Mitogen (phytohaemagglutinin, 5 µg/ml)-induced PBL proliferation was sustained on contact with isolated islets but abrogated by AEC, conditioned medium, and the islet/AEC constructs. Immunocytochemical analysis of AEC monocultures identified a subpopulation of cells that expressed the proapoptosis protein Fas ligand. This study demonstrates that human islet/AEC constructs exhibit localized immunosuppressive properties with no impairment of ß-cell function. The data suggest that transplanted islets may benefit from the immune privilege status conferred on them as a consequence of their close proximity to human AEC. Such an approach may reduce the need for chronic systemic immunosuppression, thus making islet transplantation a more attractive treatment option for the management of insulin-dependent diabetes.

Influence of microbial antigen formulation and delivery route on the immune response

Recent technological advances have resulted in the production of safe subunit and synthetic small peptide vaccines. Unfortunately, these vaccines are weakly or non-immunogenic in the absence of an immunological adjuvant (agents that can induce strong immunity to antigens). In addition, in order to prevent and/or control infection at the mucosal surface, stimulation of the mucosal immune system is essential. This may be achieved via the common mucosal immune system by exposure to antigen at a mucosal surface remote from the area of infection. Initial studies investigated the potential of multiple emulsions in effecting oral absorption and the subsequent immune responses to a lipopolysaccharide vaccine (LPS) after immunisation. Nasal delivery of LPS was carried out in parallel work using either aqueous solution or gel formulations. Tetanus toxoid vaccine in simple solution was delivered to guinea pigs as free antigen or entrapped in DSPC liposomes. In addition, adsorbed tetanus toxoid vaccine was delivered nasally free or in an aerosil gel formulation. This work was extended to investigate guinea pigs immunised by various mucosal routes with a herpes simplex virus subunit vaccine prepared from virus infected cells and delivered in gels, multiple emulsions and liposomes. Comparable serum antibody responses resulted but failed to produce enhanced protection against vaginal challenge when compared to subcutaneous immunisation with alhydrogel adjuvanted vaccine. Thus, immunisation of the mucosal surface by these methods may have been inadequate. These studies were extended in an attempt to protect against HSV genital challenge by construction of an attenuated Salmonella typhimurium HWSH aroA mutant expressing a cloned glycoprotein D-l gene fused to the Es-cherichia coli lac z promoter. Preliminary work on the colonisation of guinea pigs with S. typhimurium HWSH aroA mutants were carried out, with the aim of using the guinea pig HSV vaginal model to investigate protection.

Peptidoglycan derived from Staphylococcus epidermidis induces Connexin43 hemichannel activity with consequences on the innate immune response in endothelial cells

Gram-positive bacterial cell wall components including PGN (peptidoglycan) elicit a potent pro-inflammatory response in diverse cell types, including endothelial cells, by activating TLR2 (Toll-like receptor 2) signalling. The functional integrity of the endothelium is under the influence of a network of gap junction intercellular communication channels composed of Cxs (connexins) that also form hemichannels, signalling conduits that are implicated in ATP release and purinergic signalling. PGN modulates Cx expression in a variety of cell types, yet effects in endothelial cells remain unresolved. Using the endothelial cell line b.End5, a 6 h challenge with PGN induced IL-6 (interleukin 6), TLR2 and Cx43 mRNA expression that was associated with enhanced Cx43 protein expression and gap junction coupling. Cx43 hemichannel activity, measured by ATP release from the cells, was induced following 15 min of exposure to PGN. Inhibition of hemichannel activity with carbenoxolone or apyrase prevented induction of IL-6 and TLR2 mRNA expression by PGN, but had no effect on Cx43 mRNA expression levels. In contrast, knockdown of TLR2 expression had no effect on PGN-induced hemichannel activity, but reduced the level of TLR2 and Cx43 mRNA expression following 6 h of PGN challenge. PGN also acutely induced hemichannel activity in HeLa cells transfected to express Cx43, but had no effect in Cx43-deficient HeLa OHIO cells. All ATP responses were blocked with Cx-specific channel blockers. We conclude that acute Cx43 hemichannel signalling plays a role in the initiation of early innate immune responses in the endothelium.

Assessment of different formulations of oral Mycobacterium bovis Bacille Calmette-Guerin (BCG) vaccine in rodent models for immunogenicity and protection against aerosol challenge with M-bovis

Bovine tuberculosis (bTB) caused by infection with Mycobacterium bovis is causing considerable economic loss to farmers and Government in the United Kingdom as its incidence is increasing. Efforts to control bTB in the UK are hampered by the infection in Eurasian badgers (Metes metes) that represent a wildlife reservoir and source of recurrent M. bovis exposure to cattle. Vaccination of badgers with the human TB vaccine, M. bovis Bacille Calmette-Guerin (BCG), in oral bait represents a possible disease control tool and holds the best prospect for reaching badger populations over a wide geographical area. Using mouse and guinea pig models, we evaluated the immunogenicity and protective efficacy, respectively, of candidate badger oral vaccines based on formulation of BCG in lipid matrix, alginate beads, or a novel microcapsular hybrid of both lipid and alginate. Two different oral doses of BCG were evaluated in each formulation for their protective efficacy in guinea pigs, while a single dose was evaluated in mice. In mice, significant immune responses (based on lymphocyte proliferation and expression of IFN-gamma) were only seen with the lipid matrix and the lipid in alginate microcapsular formulation, corresponding to the isolation of viable BCG from alimentary tract lymph nodes. In guinea pigs, only BCG formulated in lipid matrix conferred protection to the spleen and lungs following aerosol route challenge with M. bovis. Protection was seen with delivery doses in the range 10(6)-10(7) CFU, although this was more consistent in the spleen at the higher dose. No protection in terms of organ CFU was seen with BCG administered in alginate beads or in lipid in alginate microcapsules, although 10(7) in the latter formulation conferred protection in terms of increasing body weight after challenge and a smaller lung to body weight ratio at necropsy. These results highlight the potential for lipid, rather than alginate, -based vaccine formulations as suitable delivery vehicles for an oral BCG vaccine in badgers.

The macrophage and the apoptotic cell:an innate immune interaction viewed simplistically?

Macrophages play important roles in the clearance of dying and dead cells. Typically, and perhaps simplistically, they are viewed as the professional phagocytes of apoptotic cells. Clearance by macrophages of cells undergoing apoptosis is a non-phlogistic phenomenon which is often associated with actively anti-inflammatory phagocyte responses. By contrast, macrophage responses to necrotic cells, including secondarily necrotic cells derived from uncleared apoptotic cells, are perceived as proinflammatory. Indeed, persistence of apoptotic cells as a result of defective apoptotic-cell clearance has been found to be associated with the pathogenesis of autoimmune disease. Here we review the mechanisms by which macrophages interact with, and respond to, apoptotic cells. We suggest that macrophages are especially important in clearing cells at sites of histologically visible, high-rate apoptosis and that, otherwise, apoptotic cells are removed largely by non-macrophage neighbours. We challenge the view that necrotic cells, including persistent apoptotic cells are, of necessity, proinflammatory and immunostimulatory and suggest that, under appropriate circumstances, persistent apoptotic cells can provide a prolonged anti-inflammatory stimulus.