Plenary Session: Plenary Session 2: The environment and autoimmunity

PBC is distinguished by immune mediated destruction of the intra hepatic bile ducts and the presence of high titer antimitochondrial autoantibodies directed against a highly specific epitope within the lipoic acid binding domain of the pyruvate dehydrogenase E2 subunit. We postulate that chemical xenobiotics modification of the lipoyl domain of PDC-E2 is sufficient to break self-tolerance, with subsequent production of AMA in patients with PBC. Indeed, using quantitative structure activity relationship (QSAR) analysis on a peptide-xenobiotic conjugate microarray platform, we have demonstrated that when the lipoyl domain of PDC-E2 was modified with specific synthetic small molecule lipoyl mimics, the ensuing structures displayed highly specific reactivity to PBC sera, at levels often higher than the native PDC-E2 molecule. Hereby, we discuss our recent QSAR analysis data on specific AMA reactivity against a focused panel of lipoic acid mimic in which the lipoyl di-sulfide bond are modified. Furthermore, data on the immunological characterization of antigen and Ig isotype specificities against one such lipoic acid mimic; 6,8-bis(acetylthio)octanoic acid (SAc), when compared with rPDC-E2, strongly support a xenobiotic etiology in PBC. This observation is of particular significance in that approximately one third of patients who have taken excessive acetaminophen (APAP) developed AMA with same specificity as patients with PBC, suggesting that the lipoic domain are a target of APAP electrophilic metabolites such as NAPQI. We submit that in genetically susceptible hosts, electrophilic modification of lipoic acid in PDC-E2 by acetaminophen or similar drugs can facilitate loss of tolerance and lead to PBC.

The Mosaic of Autoimmunity - that seemingly countless diversity in symptoms and disease presentation between individual autoimmune patients - has long seemed intractable. However, the past few years have seen an explosion in Molecular Biology's understanding of the complexity of the human body. The Human Interactome is now the name given to the map of all the interactions between metabolites of the human body. It contains all the proteins (at least 100,000), all the enzymes, peptides and steroids. We can now add to that the thousands of RNAi pathways which allow RNA to interfere with both genomic and non-genomic functions. But one question still remains: 'Why is there a Mosaic - Why is there such variability in disease presentation?' The new science of Metagenomics explains that variability between individuals. We now know the human body is a superorganism * comprising not just the 25,000 genes of Homo Sapiens, but also a million or more genes from the microbes which accumulate in our tissues as we go through life. Our mother's breast milk contains at least 400 distinct species of bacteria to seed each individual's microbiome. Throughout life we continue to accumulate microbes from our family, our environment, from our food, and even from the air we breathe. Some of these microbes have the ability to persist inside the cells of our tissues, and some can even survive inside the cells of our immune system itself. While common pathogens such as Mycobacteria and EBV have long been known to be obligate intracellular persisters, the DNA techniques developed for Metagenomics have shown us that the Human Microbiome consists of thousands, perhaps hundreds of thousands, of persistant microbial species. Not all of them are 'friendly.' Yet all of them can reduce the ability of our cells to operate correctly - they affect our Interactome - and ultimately produce that mosaic of dysfunction we know as 'autoimmunity.'

Atherosclerosis is a multifactorial chronic inflammatory disease, advanced by infectious/innate or auto-immunity accelerating mechanisms. Several epidemiological studies have revealed that host immune responses against persistent infectious pathogens, such as C. pneumonia, P. gingivalis, H. pylori, and cytomegalovirus, may promote the development of atherosclerosis. Notably, one mechanism is that immune responses against pathogen-derived heat-shock proteins that subsequently cross-react with host-derived ones contribute to atherogenesis. Another autoimmune-associated atherogenic mechanism involves autoantibodies against β2-glycoprotein I (β2GPI). β2GPI displays pleiotropic roles by influencing fibrinolysis, angiogenesis, and more recently, atherogenesis. In fact, oxLDL/β2GPI complexes formed in arterial lesions can generate an adaptive immune response (anti-oxLDL and anti-β2GPI antibodies) that up-regulate the macrophage expression of FcγRI and CD36 resulting in an increased uptake and intracellular lipid accumulation. It was also confirmed that unlike free β2GPI, oxLDL/β2GPI complexes were quickly incorporated into macrophage lysosomes where pro-inflammatory inflammasome/IL-1 responses are generated, suggesting a role of β2GPI in innate immunity and chronic inflammation. Similar to adaptive immunity, dysregulation of the innate immune response may cause tissue injury, chronic inflammation and disease. Inflammasomes are multi-molecular cytoplasmic complexes of NLRPs that regulate pro-inflammatory caspases and IL-1 in response to endogenous danger signals. Cholesterol crystals and oxidation specific epitopes (oxLDL) can activate endogenous NLRP3 inflammasomes to generate pro-inflammatory IL-1β. Thus, an inflammasome-induced IL-1β overproduction may represent an early atherogenic mechanism that initiates atherosclerosis and, OxLDL and oxLDL/β2GPI complexes may not only participate in early atherogenesis via the innate inflammasome/IL-1 pathway, but also perpetuate atherogenesis via an adaptive autoimmune response.

Systemic Lupus Erythematosus (SLE) is a chronic condition initiated by the loss of immunological tolerance to self-antigens. The pathogenic hypothesis comprises a complex interaction between genetic, environmental and hormonal factors that interact in an individual over time generating a dysregulation of the immune system leading to disease development. However, only few genes have been implicated in the contribution to SLE susceptibility. From one side, recent evidences suggest that a susceptible individual develops autoantibodies over a long time lapse due to triggering factors. Nonetheless, such autoantibody production is genetically determined and finally, the presence of autoantibodies can possibly determine the clinical presentation of SLE. Thus, the genetic predisposition to the development of autoantibodies and toward the disease process may overlap. In this light, the TRAF3IP2 gene, associated with susceptibility to psoriatic arthritis and psoriasis, encoding for Act1, has been found to be associated with disease susceptibility, with the occurrence of anti-SSB/La antibodies and with pericarditis. Since this molecule is a negative regulator of adaptive immunity and a positive signaling adaptor in IL-17 mediated immune responses, it is clear the contribution of both the innate and adaptive arms of the immune system in establishing SLE risk. Unraveling these mechanisms will allow to diagnose, to treat and even to prevent the development of SLE.

A flare of SLE disease activity can be conceptualized as the result of events at a genetic level involving several genes, which act through various mediators at a cellular level to produce the clinical phenotypes of a SLE flare. Flares of SLE disease activity are common, ranging from 0.65 to 3.0 flares per patient year in reported series, and occur in approximately 80% of patients during the course of their disease. The morbidity and mortality associated with flares can be substantial, and is related to organ damage resulting from active SLE per se and the adverse effects of corticosteroids and immunosuppressive drugs. For example, doubling of serum creatinine was 7 times more likely in patients with a renal flare, and 27 times more likely in severe renal flares manifesting as nephritic syndrome. Flares also result in significant mortality, accounting for 26% of deaths within 5 years of diagnosis and 10% of deaths more than 5 years after diagnosis in one series. Prediction of flares is thus an important goal in the management of SLE. However existing clinical and laboratory parameters do not predict flares with sufficient accuracy for clinical use, and two randomised controlled trials of corticosteroid use in patients with rising double stranded DNA levels or falling complement levels / rising complement split product levels yielded disappointing results. This highlights the needs for better markers of flares in SLE.

Kidney diseases in SLE are not always immune-complex glomerulonephritis or Òlupus nephritisÓ. Rising of serum creatinine, microhematuria and proteinuria are non-specific manifestations. Rising of serum creatinine could occur from drug-induced nephrotoxicity. Microhematuria could be found in kidney calculi or infections. Proteinuria could be caused by non-SLE glomerulopathy. Therefore, physicians have to make a differential diagnosis and provide different treatment.

Recent advances in molecular diagnostic technologies have provided new tools in diagnosis and prognosis of lupus nephritis. Study of both innate and adaptive immune responses provides novel biomarkers of lupus disease activity. Blood leucocytes from patients with active lupus nephritis were useful for diagnosis and prognostication. Myeloid-related proteins are interesting markers of innate immune response that associated with active lupus nephritis. Serum levels of B-cell signatures, like BAFF or APRIL, had been good markers and, perhaps, been targeted for therapy for SLE and nephritis.

The study of molecular signals from urinary cellular components is a logical approach in diagnosing an early relapse of nephritis and monitoring response to therapy. Urinary cytokines like interferon-inducible protein-10 (IP-10) and growth factors like vascular endothelial growth factor (VEGF) in LN are associated with the severity of renal pathology. Furthermore, the cytokine mRNAs of urine cells has been shown to be associated with response to therapy. It is likely that early treatment based on urinary molecular signals may alter the course of the disease. This would support the practicing of individualized medicine in the near future.

Along with renal damage, neuropsychiatric and cardiovascular damage has been impacting negatively on the survival of patients with systemic lupus erythematosus (SLE) for the past few decades. Discrepantly, the substantial advance in the knowledge of classification, diagnosis, therapeutic paradigm and prognosis of lupus glomerulonephritis has not been equally observed in neuropsychiatric SLE (NPSLE) and cardiovascular involvement of lupus. The limited accessibility of neural and vascular tissues for histopathological studies is probably one of the major obstacles to advance the understanding of the pathogenetic mechanism of NPSLE and cardiovascular disease in SLE. Fortunately, the recent advent of functional magnetic resonance brain imaging and biophysical endothelial function assessment has brought researchers non-invasive and sensitive approaches to unravel the functional brain and vascular pathology of lupus patients respectively, without risking the latter for unnecessary invasive procedures. With proper standardization and validation of these novel and promising tools amongst research centres, such biophysical platforms which detect organ injury with a pathogenesis-driven approach will likely add to the armamentarium of diagnostic and prognostic biomarkers for early lupus-related organ damage, and facilitate the development of potential therapeutic targets for lupus. Without these, it would be rather difficult to further improve the well-being and survival of patients with SLE.

Systemic lupus erythematosus (SLE) is an autoimmune disease that affects predominantly young women leading to significant morbidity and mortality. Neurological involvement in SLE may affect the central and peripheral nervous systems and may present with a wide variety of manifestations from headache to seizure. The underlying pathogenesis is not as well established as other manifestations of SLE and is likely heterogeneous. Diffuse neurological involvement often carries poor prognosis. The American College of Rheumatology has set up a task force in 2000 and proposed 19 classifications of neurological manifestations in SLE in order to standardize the nomenclature that may facilitate research into the underlying aetiology and treatment of this condition.

Infections are an important cause of morbidity and mortality in systemic lupus erythematosus (SLE). There are several speculations on the contributory role of microbes in the induction and reactivation of SLE largely invoking the mechanism of molecular mimicry. Susceptibility to infections in lupus may be explained by several intrinsic and acquired defects in the immune system - related to the disease itself, or to the immunosuppressive therapies. A broad spectrum of infections have been reported in SLE, including bacterial, mycobacterial, viral, fungal and parasitic infections with the respiratory and urinary tract as the most commonly involved sites. Opportunistic infections are likewise emerging as important causes of mortality. Endemicity plays an important role in augmenting susceptibility to certain infections like Salmonella and Mycobacterium tuberculosis infections particularly in developing countries.

IgA is the most abundant immunoglobulin in the human body, and performs a very specialized role which involves mucosal immunity, development of tolerance and protection against infection. IgA is the key immunoglobulin in the respiratory and gastrointestinal tracts, which provide the most intimate interface between the environment and self. Normal levels of IgA are based on early studies consisting of only small numbers of patients. The international consensus definition of IgA deficiency is a level of 0.07 g/ml after the age of four years in the absence of IgG and IgM deficiency. The epidemiology of IgA deficiency reveals interesting variances between geographical regions * the incidence in Caucasians being much higher than that in Asians. IgA deficiency has also been found to co-exist with autoimmune diseases, allergies and malignancies. The association with autoimmunity is particularly interesting because it suggests a common genetic linkage that could potentially also explain the diversity in geoepidemiology. Both MHC and non-MHC associations have been described and the 8.1 haplotype has been significantly associated with autoimmunity in IgA deficiency patients over controls. Non-MHC genetic associations include IFIH1 and CLEC16A. The mutations leading to IgA deficiency have not been defined, but in some cases of IgA deficiency it has been suggested that the pathogenesis involves a failure in switched memory B cells that can lead to this cohort experiencing an increased incidence of recurrent bacterial infections or autoimmune diseases. Attempts to investigate the role of cytokines that can induce IgA synthesis in cells of patients with IgA deficiency, such as IL21 or the combination of CD40L/anti-CD40, IL-4 and IL10, are underway.

It has been recently established that autoantibodies directed to β2-glycoprotein I (β2GPI) from patients with antiphospholipid syndrome increase the risk for atherosclerotic disease. The premature development of atherothrombosis may be explained by IgG immune complexes of β2GPI/oxidized LDL (oxLDL) that are avidly taken up by macrophages via Fcγ receptors, leading to the formation of laden-foam cells. The possible pathological mechanism has supported our recent studies: 1) Atherosclerotic plaques in arterial tissue section taken from LDL^-/- mice and WHHL rabbits were visualized by a novel imaging mass microscope (iMS) detecting lipid ligands for β2GPI. 2) Sudan IV positive-atherosclerotic lesions can be visualized by an in vivo PET/CT imaging using an anti-β2GPI scFv (lacking the Fc region; 3H3-Fv). Another set of observations using PET suggests that ectopic angiogenesis can be also detected using a β2GPI variant probe. 3) From the clinical point of view, oxLDL/β2GPI complexes and antibodies to the complexes frequently appear in patients with autoimmune, diabetes and/or cardiovascular disease (CVD), associated with vascular complications implicating β2GPI/oxLDL complexes as relevant atherogenic antigens. Current evidence points toward the atherosclerotic lesions as the primary site of oxLDL/β2GPI complex formation with subsequent targeted by a humoral immune surveillance. Finally, we report progress in the development of a novel medical system, namely, ÒTheranosticsÓ (a portmanteau of therapeutics and diagnostics) using a radiolabeled 3H3-scFv (humanized) bearing drug-containing microparticles. Thus, appropriate immunologic PET probes seem not only to be feasible but of great impact in diagnosis, prevention and management of autoimmunity and CVD.

Mann-Whitney test was used for all 2 sample comparisons(SPSS, version 13.0). P<0.05 was considered significant.