Faculty of Medicine,Dentistry and Health Sciences Department of Pathology

John Underwood

Contact: John Underwood
Phone: +61 3 8344 4292
Fax: +61 3 8344 4004
Email: johnru@unimelb.edu.au


Key Research Programs and Objectives:

Investigations of Autoimmunity in Health and Disease:

Using and experimental mouse model, our studies investigating autoimmunity in health and disease were amongst the first to demonstrate that autoreactive B lymphocytes constituted a significant component of the normal immune repertoire of healthy mice. These studies indicated the diversity of the autoantibodies produced by the autoreactive immune repertoire and posed the question of the relationship of this reactivity to autoantibody production in autoimmune diseases. These studies have been extended to the molecular analysis of the antigen combining sites of autoantibodies from healthy mice and their comparison to antibodies of similar specificities derived from autoimmune diseases.

The observation that autoreactive B lymphocytes constitute a significant component of the normal immune repertoire poses questions as to the functional basis of the persistence of such cells in the healthy animal. This study has been extended to encompass molecular investigations of the autoreactive B lymphocyte repertoire of healthy animals in order to define the proportions of autoreactive B lymphocytes and the specificities and functions of the corresponding autoantibodies in healthy animals in relation to counterpart antibodies in autoimmune diseases. Our initial investigations of over 8000 B lymphocyte hybridomas derived from healthy mice, revealed that approximately 10% of the normal B lymphocyte repertoire of healthy mice is committed to the production of autoantibodies. This autoreactive B lymphocyte repertoire analysis showed that the majority of the B lymphocytes identified from healthy animals produced autoantibodies to intracellular autoantigens which are also identified by autoantibodies derived from a variety of human and murine autoimmune diseases. One of the hypotheses under examination is whether the B lymphocytes isolated from healthy animals are the precursors of B lymphocytes which secrete autoantibodies in autoimmune diseases. Immunocytochemical, immunochemical, epitope mapping and immunoglobulin V H and V L domain sequencing techniques are being used to resolve this question.

Consistent with autoantibodies derived from many autoimmune diseases, investigation of the antigenic specificities of the autoantibodies isolated from the unimmunized, healthy mice has revealed the ability of these antibodies to react with multiple, apparently unrelated self and non-self antigens (antibody polyspecificity). To elucidate the molecular basis of antibody polyspecificity, polyspecificity profiles of 19 autoantibodies exhibiting reactivity to five groups of autoantigens have been defined on a panel of 23 homologous and heterologous proteins, phospholipids, haptens and bacterial antigens using indirect and inhibition ELISAs. From immunoassay analysis, each of the 19 monoclonal autoantibodies derived from the unimmunized, healthy mice have been found to exhibit unique profiles of reactivity on the 23 antigen panel. Comparison of the sequences of the V H and V L domains of selected polyspecific autoantibodies is aimed at determining whether reactivity to overlapping to groups of antigens is mediated via interactions with antigen binding subsites within the complementarity determining regions (CDRs) of the corresponding immunoglobulins. Preliminary evidence indicates that polyspecific recognition of overlapping sets of identical antigens by each of the anti-intermediate filament and anti-nuclear autoantibodies occurs via different CDR sequences within the corresponding V H and V L antibody domains. These observations implicate antibody polyspecificity as an additional mechanism for amplifying existing immune diversity within the B lymphocyte repertoire.

As unique polyspecificity profiles have been defined for the 19 autoantibodies examined in this study, the working hypothesis is that the observed variations in polyspecificity profiles represent a means for identifying differences in antigen combining site structures between antibodies. Validation of this hypothesis would indicate that such profiles provide another method of comparing the antigen combining sites of polyspecific autoantibodies derived from health and disease.

In an attempt to further investigate of the molecular basis of antibody polyspecificity, we are currently undertaking collaborative X-ray crystallographic studies of the antigen combining sites of polyspecific autoantibodies with Dr Paul Ramsland at the Austin Research Institute. These studies involve the comparative analysis of Fab fragments generated from monoclonal antibodies exhibiting unique polyspecificity profiles.

Anti-idiotype antibody suppression of cytotoxic pre-and post-transplant anti-HLA antibodies

Organ transplantation is now recognized as one of the most common therapies for kidney, liver, heart and lung organ failure. The number of patients awaiting transplants for damaged or dysfunctional organs greatly exceeds compatible donor organ numbers due to the shortage of human donor organs. Graft rejection is an inherent complication of transplantation. Immunosuppressive drugs and anti-T lymphocyte antibodies can prevent T lymphocyte-mediated acute and chronic graft rejection. Cytotoxic anti-HLA antibodies also represent a major problem in solid organ transplantation. Pre-existing cytotoxic anti-HLA antibodies prevent patient access to transplant therapy and such antibodies arising post-transplantation correlate with graft rejection and poor prognosis.

Intravenous Immunoglobulin (IVIg), representing IgG from pooled plasma of several thousand healthy blood donors, is not only an essential substitutive therapy for immunodeficiency syndromes but also an important immunomodulatory therapy for autoimmune and systemic inflammatory diseases and in bone marrow and solid organ transplantation. IVIg contains antibodies to infectious microorganisms and non-infectious, non-self antigens, natural autoantibodies and immunoregulatory anti-idiotype, anti-cell surface and anti-cytokine antibodies. Anti-idiotype and anti-cytokine antibodies, Fc receptor blockade and interference with cellular activation by antibodies to lymphocyte cell membrane signal transduction molecules are proposed as mechanisms by which IVIg regulates autoimmune and systemic inflammatory diseases and maintains of immunological tolerance.

In solid organ transplantation, the immunomodulatory efficacy of IVIg is dependent on anti-idiotype antibody-mediated inhibition of cytotoxic anti-HLA antibodies. The variability in the efficacy of different IVIg preparations for ameliorating anti-HLA antibody cytotoxicity reflects differences in the regulatory immunoglobulin content. This new collaboration between Associate Professor Brian Tait of the Victorian Transplantation and Immunogenetics Service, ARCBS and Dr John Underwood of Pathology is aimed at the production of defined and highly specific anti-idiotype antibodies reactive with common anti-HLA antibody specificities would provide a reproducible and safe immunoregulatory therapy in solid organ transplantation.Such anti-idiotype antibody reagents would substantially reduce health care costs by reducing ongoing health care associated with delays in identification of compatible transplant donors and would eliminate the need for expensive and complex immunosuppressive therapies in patients undergoing anti-HLA antibody-mediated acute and chronic transplant rejection and the associated health care costs associated with the side-effects of these therapies.

Investigation of Immunomodulatory Botanical Medicinals

Over 64% of the World’s population uses plant-derived traditional medicines for primary health care. In addition, over 100 of the most commonly used drugs in the developed World such as asprin, digoxin, morphine and taxol were originally derived from plants. Although many traditional herbal medicines have been used for hundreds or thousands of years, one of the confounding issues with their use as Complementary or Alternative medicines in modern society is the lack of rigorous scientific investigation of the bioactive components, cellular and molecular mechanisms of action and clinical efficacy or otherwise of these medicines. The objective of our laboratory is to scientifically examine the immunomodulatory properties of a range of traditional herbal medicines with a view to the use of these plant-derived products as direct or indirect immune stimulators. Our initial studies were aimed at the investigation of the immunomodulatory properties of selected herbal medicines to defined experimental and natural antigens in an in vivo animal model. Following on from these studies, in vitro experiments are being used to determine the cellular and molecular bases of the observed immunomodulatory responses. Future studies will be aimed at the identification, isolation and chemical characterization of the bioactive components of plants responsible for initiating the observed immunostimulation.

A number of adverse reactions have been reported following the use of herbal medicines. Ongoing studies in this laboratory have investigated the potential for the induction of adverse reactions following administration of selected herbal medicines including the production of organ-specific and non-organ-specific autoantibodies, inflammation and microstructural changes in host tissues.

The key objectives of these studies are to identify plant constituents that can initiate and maintain long term stimulation of the host immune system thereby providing enhanced protection against viral, bacterial and parasitic diseases when used in conjunction with conventional or modified immunization strategies. A high proportion of the World’s population live in developing countries and rely on traditional medicines for primary health care. One long term vision of this research is that following the validation of the efficacy of the plant-derived immunostimulatory substances, developing countries could build up industries “in country” based on the cultivation, harvesting, processing, marketing and distribution of plant-derived traditional medicines. Such industries would provide not only improved health outcomes but also the added benefits of increased employment and economic gains.

Integration of Teaching and Research

An ongoing commitment in the Underwood laboratory is the integration of research with the scientific training of undergraduate and post-graduate students. To date 36 B.Sc. Honours students and 15 post-graduate students, including four M.Sc. students and 12 Ph.D. students have been supervised by Dr Underwood. Post-graduate students emerging from this laboratory have taken up key positions in teaching, major international pharmaceutical and biotechnology companies, research institutes and government departments involved in funding novel biotechnology initiatives.

Techniques:

The following techniques are routinely used in the laboratory:

Collaborations

Departmental:

External:

International:

Funding:

Support is available from industry-based sources

Selected Publications:

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