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Biomedical subjects

A M Mowat

Publications and source records attributed to A M Mowat.

At least 19 recordsLinked to original sources
PubMed2001

CTA1-DD-immune stimulating complexes: a novel, rationally designed combined mucosal vaccine adjuvant effective with nanogram doses of antigen.

Mucosally active vaccine adjuvants that will prime a full range of local and systemic immune responses against defined antigenic epitopes are much needed. Cholera toxin and lipophilic immune stimulating complexes (ISCOMS) containing Quil A can both act as adjuvants for orally administered Ags, possibly by targeting different APCs. Recently, we have been successful in separating the adjuvant and toxic effects of cholera toxin by constructing a gene fusion protein, CTA1-DD, that combines the enzymatically active CTA1-subunit with a B cell-targeting moiety, D, derived from Staphylococcus aureus protein A. Here we have extended this work by combining CTA1-DD with ISCOMS, which normally target dendritic cells and/or macrophages. ISCOMS containing a fusion protein comprising the OVA(323-339) peptide epitope linked to CTA1-DD were highly immunogenic when given in nanogram doses by the s.c., oral, or nasal routes, inducing a wide range of T cell-dependent immune responses. In contrast, ISCOMS containing the enzymatically inactive CTA1-R7K-DD mutant protein were much less effective, indicating that at least part of the activity of the combined vector requires the ADP-ribosylating property of CTA1. No toxicity was observed by any route. To our knowledge, this is the first report on the successful combination of two mechanistically different principles of adjuvant action. We conclude that rationally designed vectors consisting of CTA1-DD and ISCOMS may provide a novel strategy for the generation of potent and safe mucosal vaccines.

Adjuvants, Immunologic↗
PubMed2001

Oral tolerance.

The ability of the mucosal immune system to distinguish between harmful and harmless antigens is essential for mounting protective immune responses and preventing the induction of mucosal pathology yet the basis for this remains unclear. As fed antigen can also exert systemic effects understanding oral tolerance and priming will also have important consequences for therapy and vaccination. Here we will not only review the increasing amount of information about the potential mechanisms of oral tolerance and priming but also attempt to shed some light on how differences in the uptake and handling i.e. 'the journey' of orally administered antigen may promote these mechanisms.

Animals↗
PubMed2000

Induction of oral tolerance in the primed immune system: influence of antigen persistence and adjuvant form.

Oral tolerance is being promoted as a therapy for autoimmune diseases and therefore will need to be functional in a primed immune system. In previous studies, we found that although primed mice could be tolerized by feeding ovalbumin (OVA), the degree of the tolerance and its effects on individual components of the systemic immune response were more limited than that found in naive animals. Here we increased the dose and frequency of antigen feeding in an attempt to extend the effects of oral tolerance in primed mice and to understand why its effects are limited under these conditions. Increasing the amounts of OVA fed, up to a single dose of 400 mg, or using multiple feeds of 5 x 5 or 5 x 25 mg OVA, did not radically alter the extent of tolerance, with DTH responses, antigen-specific proliferation, and IL5 and IFN-gamma production still being tolerized, but antibody responses remaining generally resistant. The deficient tolerance in primed mice could not be overcome by waiting for maximum clonal expansion to wane and was not influenced by persistent release of antigen from a depot adjuvant. We conclude that the resistance of primed mice to oral tolerance may be due to the fact that antigen-experienced T cells may be inherently resistant to induction of tolerance, or that the microenvironment of the primed immune system inhibits the delivery of tolerogenic signals to antigen-specific T cells.

Administration, Oral↗
PubMed1999

Normal induction of oral tolerance in the absence of a functional IL-12-dependent IFN-gamma signaling pathway.

There is considerable evidence that regulatory cytokines play an important role in mediating the systemic tolerance found after oral administration of protein Ags. Although most existing work has focused on cytokines such as IL-4, IL-10, and TGF-beta, recent evidence from TCR transgenic systems suggests that the induction of oral tolerance is accompanied by priming of Ag-specific IFN-gamma production. IFN-gamma has also been implicated as a mediator of T cell tolerance in other models in vivo and in vitro, including that induced by aerosol administration of protein. We show here that feeding tolerogenic doses of OVA primes for IFN-gamma production in the spleen of mice with a normal T cell repertoire. However, depleting IFN-gamma at the time of feeding OVA had no effect on the induction of tolerance. In addition, tolerance was induced normally in both IFN-gamma receptor knockout (IFN-gammaR-/-) and IL-12 p40 knockout (IL-12-/-) mice. This was the case for all components of the systemic immune response and also with a variety of feeding protocols, including those believed to induce distinct regulatory mechanisms. We conclude that IL-12-dependent IFN-gamma-mediated regulation does not play an essential role in oral tolerance.

Administration, Oral↗
PubMed1999

Immune-stimulating complexes induce an IL-12-dependent cascade of innate immune responses.

The development of subunit vaccines requires the use of adjuvants that act by stimulating components of the innate immune response. Immune-stimulating complexes (ISCOMS) containing the saponin adjuvant Quil A are potential vaccine vectors that induce a wide range of Ag-specific responses in vivo encompassing both humoral and CD4 and CD8 cell-mediated immune responses. ISCOMS are active by both parenteral and mucosal routes, but the basis for their adjuvant properties is unknown. Here we have investigated the ability of ISCOMS to recruit and activate innate immune responses as measured in peritoneal exudate cells. The i.p. injection of ISCOMS induced intense local inflammation, with early recruitment of neutrophils and mast cells followed by macrophages, dendritic cells, and lymphocytes. Many of the recruited cells had phenotypic evidence of activation and secreted a number of inflammatory mediators, including nitric oxide, reactive oxygen intermediates, IL-1, IL-6, IL-12, and IFN-gamma. Of the factors that we investigated further only IL-12 appeared to be essential for the immunogenicity of ISCOMS, as IL-6- and inducible nitric oxide synthase knockout (KO) mice developed normal immune responses to OVA in ISCOMS, whereas these responses were markedly reduced in IL-12KO mice. The recruitment of peritoneal exudate cells following an injection of ISCOMS was impaired in IL-12KO mice, indicating a role for IL-12 in establishing the proinflammatory cascade. Thus, ISCOMS prime Ag-specific immune responses at least in part by activating IL-12-dependent aspects of the innate immune system.

Adjuvants, Immunologic↗
PubMed1999

Oral vaccination with immune stimulating complexes.

There is a need for non-living adjuvant vectors which will induce a full range of local and systemic immune responses to orally administered purified antigens. Here we describe our experience with lipophilic immune stimulating complexes (ISCOMS) containing the saponin adjuvant Quil A. When given orally, ISCOMS containing the model protein antigen ovalbumin (OVA) induce a wide range of systemic immune responses, including Th1 and Th2 CD4 dependent activity, class I MHC restricted cytotoxic T-cell responses and local production of secretory IgA antibodies. More recent results indicate that ISCOMS may act partly by enhancing the uptake of protein from the gut. In addition, intraperitoneal injection of ISCOMS recruits and activates many components of the innate immune system. including neutrophils, macrophages, and dendritic cells. In parallel, there is increased production of nitric oxide (NO), reactive oxygen intermediates (ROI), interleukins (IL) 1, 6, 12, and gamma interferon (gammaIFN). Of these factors, only IL12 is essential for the immunogenicity of ISCOMS in vivo, as mucosal and systemic responses to ISCOMS are reduced in IL12KO mice, but not in IL4KO, IL6KO, inducible NO synthase (iNOS) KO, or gammaIFN receptor KO mice. We propose that ISCOMS act by targetting antigen and adjuvant to macrophages and/or dendritic cells. This pathway may be amenable to exploitation for vaccine development, especially if combined with another vector with a different mucosal adjuvant profile, such as cholera toxin.

Adjuvants, Immunologic↗
PubMed1998

Expanding dendritic cells in vivo enhances the induction of oral tolerance.

The intestine is under perpetual challenge from both pathogens and essential nutrients, yet the mucosal immune system is able to discriminate effectively between harmful and innocuous Ags. It is likely that this selective immunoregulation is dependent on the nature of the APC at sites where gut Ags are processed and presented. Dendritic cells (DC) are considered the most potent of APC and are renowned for their immunostimulatory role in the initiation of immune responses. To investigate the role of DC in regulating the homeostatic balance between mucosal immunity and tolerance, we treated mice with Flt3 ligand (Flt3L), a growth factor that expands DC in vivo, and assessed subsequent systemic immune responsiveness using mouse models of oral tolerance. Surprisingly, mice treated with Flt3L to expand DC exhibited more profound systemic tolerance after they were fed soluble Ag. Most notably, tolerance could be induced in Flt3L-treated mice using very low doses of Ag that were ineffective in control animals. These findings contrast with the generally accepted view of DC as immunostimulatory APC and furthermore suggest a pivotal role for DC during the induction of tolerance following mucosal administration of Ag.

Administration, Oral↗
PubMed1998

Immunological consequences of intervention in established immune responses by feeding protein antigens.

The usual result of feeding protein antigens to naive animals is the induction of profound immunological unresponsiveness and this is currently being exploited to treat inflammatory disease. Because the most useful therapeutic application of feeding antigen would be to suppress established disease, the aim of this study was to compare the immunological basis of oral tolerance induced by feeding a model antigen to naive and primed animals. We show that feeding 2-200 mg ovalbumin (OVA) to mice 7 days after immunisation with OVA in adjuvant produces dose-dependent suppression of delayed-type hypersensitivity (DTH), T cell proliferation, and both TH1 and TH2 cytokines, although serum IgG levels were unaffected. Feeding OVA before immunisation suppressed all these responses. Although feeding up to 8 days after immunisation could suppress some subsequent responses, tolerance was induced much more effectively when antigen was fed in the first 4 days after immunisation. Tolerance in primed mice was intact in IL-4-/- mice, indicating that it was not caused by selective upregulation of TH2 cells in vivo. We conclude that oral administration of protein antigen can inhibit ongoing responses by all effector T cell subsets, but the exact consequences, and therefore possibly the mechanisms, are different from those induced by tolerising naive mice. These findings may have important implications for designing therapeutic regimes exploiting oral tolerance.

Animals↗
PubMed1998

Immune responses to dietary antigens: oral tolerance.

The concept of immunologically mediated tolerance to food antigens through exposure to mucosal antigen has been the subject of continuous scientific debate. After a decline in interest in the mid-1980s, oral tolerance has again attracted the attention of immunologists. Here, Stephan Strobel and Allan Mowat discuss how this central immunological principle has potential new therapeutic applications for the treatment of autoimmune, inflammatory and possibly food-allergic diseases.

Animals↗
PubMed1998

Preservation of mucosal and systemic adjuvant properties of ISCOMS in the absence of functional interleukin-4 or interferon-gamma.

Adjuvants are a critical component of non-viable vaccine vectors, particularly for those to be used via mucosal routes. Although most adjuvants act by inducing local inflammatory responses, the molecular basis of many of these effects is unclear. Here we have investigated whether interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) are required for the induction of local and systemic immune responses by oral and parenteral administration of ovalbumin (OVA) in immune stimulating complexes (ISCOMS), a potent mucosal adjuvant vector. Our results show that after oral or systemic immunization with OVA ISCOMS, IL-4 knockout (IL4KO) and IFN-gamma receptor knockout (IFN-gamma RKO) mice develop an entirely normal range of immune responses including delayed-type hypersensitivity (DTH), serum immunoglobulin G (IgG) antibodies, T-cell proliferation and cytokine production, class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocyte (CTL) activity and intestinal IgA antibodies. These responses were of a similar magnitude to those found in the wild-type mice, indicating that the immunogenicity of ISCOMS is not influenced by the presence of IL-4 or IFN-gamma and emphasizing the potential of ISCOMS as widely applicable mucosal adjuvants.

Adjuvants, Immunologic↗
PubMed1998

Immune stimulating complexes as mucosal vaccines.

There is a need for non-living adjuvant vectors that will allow a full range of local and systemic immune responses to orally administered purified antigens. Here we describe our experience with lipophilic immune-stimulating complexes (ISCOMs) containing the saponin adjuvant Quil A. When given orally, ISCOMs containing the model protein antigen ovalbumin (OVA) induce a wide range of systemic immune responses, including Th1 and Th2 CD4-dependent activity, serum IgG antibodies and class I MHC-restricted cytotoxic T cell responses. In addition, there is local production of secretory IgA antibodies in the intestine itself, as well as priming of CD4 and CD8 T cell responses in the draining lymphoid tissues. Preliminary results indicate that the mucosal adjuvant properties of ISCOMs may reflect their ability to deliver antigen combined with the pro-inflammatory properties of Quil A in a particulate form. Of the many inflammatory mediators induced, interleukin-12, derived from dendritic cells and/or macrophages, appears to be of central importance. These results indicate that ISCOMs may prove to be useful mucosal vaccine vectors with functions which are distinct from existing vectors of this type.

Animals↗
PubMed1997

Neutralizing IL-12 during induction of murine acute graft-versus-host disease polarizes the cytokine profile toward a Th2-type alloimmune response and confers long term protection from disease.

Injection of parental spleen cells into BDF1 mice results in a graft-vs-host disease (GVHD), the nature of which is critically dependent on the parental haplotype. B6-->BDF1 mice develop a Th1-mediated immunosuppressive lethal GVHD, whereas DBA/2-->BDF1 mice develop a Th2-dependent chronic GVHD, characterized by autoantibody production and glomerulonephritis. In this study we show that neutralizing endogenous IL-12 for a brief period during the initiation of acute GVHD in B6-->BDF1 mice not only confers long term protection from the acute disease, but also permits full repopulation of the recipient with donor B6 lymphocytes. Antibody-treated animals showed normal T cell proliferation in response to Con A stimulation and remained healthy throughout the study. Splenocytes from such mice showed reduced in vitro production of IFN-gamma and enhanced production of IL-5 and IL-10, suggesting a permanent switch from a Th1 to a Th2 cytokine response, comparable to that associated with chronic GVHD in DBA/2-->BDF1 mice. In contrast to DBA/2-->BDF1 mice, however, anti-IL-12-treated B6-->BDF1 mice displayed only mild B cell hyper-responsiveness, as evidenced by a modest increase in serum IgG and IgE levels and moderate levels of anti-dsDNA Abs. Importantly, however, anti-IL-12-treated B6-->BDF1 mice showed no evidence of immune complex-mediated glomerulonephritis. These results demonstrate that neutralizing IL-12 is an effective means of preventing acute GVHD and does not result in the development of chronic GVHD, which might otherwise limit its application.

Acute Disease↗
PubMed1997

The anatomical basis of intestinal immunity.

The lymphoid tissues associated with the intestine are exposed continuously to antigen and are the largest part of the immune system. Many lymphocytes are found in organised tissues such as the Peyer's patches and mesenteric lymph nodes, as well as scattered throughout the lamina propria and epithelium of the mucosa itself. These lymphocyte populations have several unusual characteristics and the intestinal immune system is functionally and anatomically distinct from other, peripheral compartments of the immune system. This review explores the anatomical and molecular basis of these differences, with particular emphasis on the factors which determine how the intestinal lymphoid tissues discriminate between harmful pathogens and antigens which are beneficial, such as food proteins or commensal bacteria. These latter antigens normally provoke immunological tolerance, and inappropriate responses to them are responsible for immunopathologies such as food hypersensitivity and inflammatory bowel disease. We describe how interactions between local immune cells, epithelial tissues and antigen-presenting cells may be critical for the induction of tolerance and the expression of active mucosal immunity. In addition, the possibility that the intestine may act as an extrathymic site for T-cell differentiation is discussed. Finally, we propose that, under physiological conditions, immune responses to food antigens and commensal bacteria are prevented by common regulatory mechanisms, in which transforming growth factor beta plays a critical role.

Animals↗
PubMed1997

Mechanisms of oral tolerance.

Oral tolerance is the specific immunological unresponsiveness induced by feeding antigen. Although it is an obstacle to oral vaccination, it is probably the mechanism that prevents intestinal hypersensitivity reactions to food antigens and may provide a novel strategy for the treatment of a range of inflammatory disorders. Feeding antigen can provide stable and long-lasting tolerance of a wide range of immune responses to a variety of antigens. However, the mechanisms of oral tolerance and the major factors that influence them remain controversial.

Administration, Oral↗