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Immune-mediated, drug-induced hepatitis is a rare complication of halogenated volatile anesthetic administration. IL-4-regulated Th2-polarized reactions initiate this type and other types of hepatitis, while the mechanisms that regulate the severity remain elusive. IL-33 is an innate, IL-4-inducing, Th2-polarizing cytokine that has been detected in patients with liver failure and has been associated with upregulated ST2+Foxp3+CD4+CD25+ T cells; however, roles for IL-33 in drug-induced hepatitis are unclear. We investigated IL-33 in an anesthetic, immune-mediated hepatitis modeled in BALB/c, IL-33−/− and ST2−/− mice, as well as in patients with anesthetic hepatitis. The hepatic IL-33 and ST2 levels were elevated in BALB/c mice (p

The disease entity today widely called ‘primary biliary cirrhosis’ was first described by Addison and Gull in 18511 and Hanot in 1876.2 One hundred years after its first description, MacMahon and Thannhauser3 proposed the term ‘xanthomatous biliary cirrhosis’ for this disease based on the typical xanthoma formation with accumulation of cholesterol esters in the skin around the eyes in association with inflammatory destruction of small intrahepatic bile ductules leading to a biliary type cirrhosis. Xanthoma formation, however, is not a very common sign in this disorder. This may be the reason why the term ‘primary biliary cirrhosis’, proposed 1 year later for the same disorder by Ahrens et al ,4 gained wider acceptance when most patients were presenting with advanced liver disease. Dame Sheila Sherlock, already in 1959, opposed the term ‘primary biliary cirrhosis’ as many of her patients were free of cirrhosis at the time of diagnosis and the mean survival was 5 and a half years (3–11) even for the fatal cases, whereas many asymptomatic patients would survive more than 10 years.5 The term ‘primary biliary cirrhosis’ remained an issue of concern as reflected by the name change proposal of Rubin, Schaffner and Popper in 1965 with their paper ‘Primary biliary cirrhosis—Chronic non-suppurative destructive cholangitis’.6 Sherlock7 wisely commented on this new name: “…a better one, although it is unlikely that it will replace the more popular, although inaccurate, one of primary biliary cirrhosis”. She was right, again. And even 40 years later, the European8 and American9 Clinical Practice Guidelines still used the term ‘primary biliary cirrhosis’ even though it was an anachronism and did not accurately reflect the natural history of disease in the vast majority of patients as it is today. The early diagnosis of primary biliary cirrhosis has …

Autoimmune hepatitis (AIH) was first studied under its earlier name of “chronic active hepatitis” (CAH) from the 1950s, coincident with a renaissance of interest in autoimmunity. The definition of autoimmune serum reactants in disease, including CAH, gave new insights into chronic hepatitis and liver cirrhosis, and led to refinements of Burnet's clonal selection theory of acquired immunity, 1957–59. Various discoveries including serological reactants in CAH prompted its designation in 1965 as autoimmune hepatitis, and treatment with immunosuppressive drug regimens transformed outcomes and survival. Serological observations further indicated that AIH could exist as either of two types, clinically similar but genetically different: Type 1 aligned more with the non-organ-specific multisystem diseases, and the infrequent Type 2 more with the organ-specific diseases. However, events in either type that could explain the onset of autoimmunity in the normally tolerogenic milieu of the liver have not been discerned. In the genetically predisposed individual, initiation may depend on non-specific death of hepatocytes after which fragments derived from disordered apoptosis acquire the capacity for ongoing auto-immunogenic stimulation. Insufficiency in numbers and function of Treg populations appears important in the promotion of this autoimmune process.

AIMS/HYPOTHESIS: Established autoimmune markers of type 1 diabetes, including islet cell autoantibodies (ICA) and autoantibodies to glutamic acid decarboxylase (GADA) have been used to screen people presenting with type 2 diabetes for latent autoimmune diabetes in adults. We have examined the prevalence of autoantibodies to protein tyrosine phosphatase isoforms IA-2 (IA-2A) and IA-2beta/phogrin (IA-2betaA) in a cohort of adult UKPDS patients thought to have type 2 diabetes, and investigated the possible role of these autoantibodies in predicting requirement for insulin therapy. METHODS: IA-2A and IA-2betaA were measured by a validated radioimmunoassay with human recombinant autoantigens in 4,169 white Caucasian patients aged 25-65 years and newly diagnosed with type 2 diabetes. The clinical requirement for insulin therapy within 6 years was examined in 2,556 patients not randomised to insulin. RESULTS: IA-2A and IA-2betaA were present in 2.2 and 1.4%, respectively, of these patients. IA-2A were more prevalent in younger patients (p for trend

Primary biliary cholangitis (PBC) has been regarded as female-predominant without evidence of gender difference in survival. We aimed to compare the overall survival, incidence and prevalence of PBC in two well defined population-based studies over a recent decade, considering also sex ratios and mortality. We have taken advantage of population-wide records, during 2000–2009, in Lombardia, Northern Italy and Denmark. We focused on the incident cases of PBC, including gender and outcome, among 9.7 million inhabitants of Lombardia and 5.5 million of Denmark. In Lombardia there were 2,970 PBC cases with a female:male ratio of 2.3:1. The age/sex-adjusted annual incidence of PBC was 16.7 per million. Point prevalence was 160 per million on January 1st 2009. In Denmark there were 722 cases of incident PBC, female:male ratio was 4.2:1 and the annual incidence was 11.4 per million, a point prevalence of 115 per million in 2009. Cox regression multivariate analysis identified male sex as an independent predictor of all-cause mortality in both Italian (HR 2.36) and Danish population (HR 3.04). Our data indicate for PBC a sex ratio significantly lower than previously cited, a reversal of the usual latitudinal difference in prevalence and a surprisingly higher overall mortality for male patients.

Summary Although the hallmark of primary biliary cirrhosis (PBC) is the presence of anti-mitochondrial antibodies (AMA), a significant number of patients have anti-nuclear antibodies (ANA) directed primarily against two nuclear proteins, gp210 and sp100. In PBC, there are considerable data on the specificity of these anti-nuclear antibodies as well as suggestive evidence that antibodies to gp210 predict a poor outcome. However, a further understanding of the significance of these autoantibodies has been hampered by limitations in accessing human subjects in a preclinical or early asymptomatic stage. To overcome this limitation, we have taken advantage of transgenic mice with abrogated transforming growth factor-β signalling in T cells (dnTGF-βRII) that develop histological features of PBC as well as the same AMA specificity. We studied these mice for serum ANA, including specific autoantibodies against gp210 and sp100. We further examined sera from dnTGF-βRII mice with concurrent deletions of the genes encoding interleukin (IL)-12p35, IL-12p40, IL-23p19, IL-17, IL-6, interferon (IFN)-γ or tumour necrosis factor (TNF)-α. Sera from all the dnTGF-βRII mouse lines contained antibodies against gp210 and sp100. Of significance, mice with germline deletions of the genes encoding IL-12p40, IL-23p19, IL-17, IL-6 and TNF-α had significantly lower titres of anti-gp210 antibodies. These results provide a platform to dissect the mechanisms of gp210 and sp100 autoantibody production in dnTGF-βRII mice as well as to study the possible role of ANA in the pathophysiology of PBC.

Autoimmune hepatitis (AIH), formerly known as chronic active hepatitis (CAH), first attracted attention around 1948. It probably existed as “subacute hepatitis” before that time, however. Young women were mainly affected and the outlook was poor. Suspicions of an immunological abnormality in CAH were raised by extreme hypergammaglobulinemia, but the likely primary culprit then seemed to be a persistently active virus in the liver. Various anti-tissue antibodies became recognized during 1955 to 1965, detected first to nuclear antigens by the lupus erythematosus (LE) cell test and immunofluorescence (IFL) for antinuclear autoantibody, then to a smooth muscle antigen with the true reactant later identified as the microfilament F actin, and then to an antigen enriched in endoplasmic reticulum (microsomes) of liver and kidney cells. The availability of recombinant or finely purified autoantigen now allows for automated molecular assays for some of these reactivities and this, with improved IFL technologies, has led to serological confidence in diagnosis with institution of highly effective suppressive therapies. Meanwhile immunologists remain sorely challenged in their attempts to define the pathogenetic steps from initiation, relentless persistence to ultimate hepatocytolytic destruction in this enigmatic liver disorder, AIH. Autoimmune hepatitis has been a controversial subject since being named as such in 1965,[6][1] with eventual international endorsement in 1993.[7][2] Hence, relevant historical material is included. * AIH : autoimmune hepatitis CAH : chronic active hepatitis LE : lupus erythematosus SLE : systemic lupus erythematosus ANA : anti-nuclear autoantibodies IFL : immunofluorescence SMA : smooth muscle antibody LKM : liver and kidney microsomes HBV : hepatitis B virus HCV : hepatitis C virus IAIHG : International Autoimmune Hepatitis Group PBC : primary biliary cirrhosis CHC : chronic hepatitis C UNL : upper normal limit Ig : immunoglobulin IgG : immunoglobulin G SLA/LP : soluble liver antigen and liver pancreas LC-1 : liver cytosol antigen type 1 IUIS : International Union of Immunological Societies ds : double-stranded ss : single-stranded ANCA : anti-neutrophil cytoplasmic antibody cANCA : cytoplasmic anti-neutrophil cytoplasmic antibody pANCA : perinuclear anti-neutrophil cytoplasmic antibody pANNA : peripheral anti-neutrophil nuclear antibody ELISA; : enzyme-linked immunosorbent assay G : globular F : filamentous LP : liver-pancreas antigen SLA : soluble liver antigen tRAP; : tRNA-associated protein SHMT : serine hydroxymethyl transferases UGT : uridine diphosphate glucuronosyl transferases APECED : autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy APS-1 : autoimmune polyendocrine type 1 FTCD : formiminotransferase cyclodeaminase LSP : liver-specific lipoprotein ASGPR : asialoglycoprotein receptor PSC : primary sclerosing cholangitis AIRE : autoimmune regulator RLN : regional lymph node [1]: #ref-6 [2]: #ref-7

Autoimmune hepatitis (AIH) as chronic active hepatitis became recognized in the 1940s as a progressive hyperglobulinemic disease affecting younger women attributed to persisting virus infection of the liver: autoimmunity then was barely on the horizon.The lupus erythematosus (LE) cell reported in 1948 signified the presence of antinuclear autoantibodies, promoting perceptions of autoimmunity in certain chronic diseases. Recognition of LE cells in chronic hepatitis led to the designation of 'lupoid hepatitis', with autoimmunity further substantiated by anti-cytoplasmic autoantibodies detected by complement fixation. Next a serum reactant with smooth muscle of rodent stomach was found to have a wider distribution and became identified as an autoantibody to filamentous (F) actin. Therapy with corticosteroids proved effective, particularly combined with azathioprine. Various trials showed greatly improved survival and established modern therapy of AIH. An HLA-based predisposition (B8, DR3) was the first pointer to a genetic etiology.Recombinant or purified autoantigenic substrates have led to automated assays, which, together with improved immunofluorescence procedures, allow serological confidence in diagnosis and institution of effective immunosuppressive therapies. The liver-kidney 'microsomal' autoantigen reactive with cytochrome P450 2D6 distinguishes two serological types of AIH that appear pathogenetically distinct. Molecular characterization of antigens and epitopes remains wanting in type 1 AIH.The challenge remains with both types of AIH to elucidate in molecular terms the genetic and environmental basis of pathogenesis from initiation to ultimate progression and cirrhosis (when inadequately treated). Advancing technologies are bringing this goal closer to being attainable.

The paradoxical relationship between immunodeficiency (under-responsiveness) and autoimmunity (over-responsiveness) affecting the immune system was debated at the Fourth AARDA Colloquium on cross-disciplinary issues in autoimmunity. Immunodeficiency disease and autoimmune disease, far from being mutually exclusive, share profound dysregulation of the immune system. Among the most keenly discussed issues were: i) the remarkably high number of molecularly identified primary immunodeficiencies with autoimmune expressions; ii) the homeostasis of immune function such that deficiency in any one given compartment can result in over activity in the same or another compartment; iii) whilst some immune deficiency states are essentially monogenic, each of them can exhibit striking variability in autoimmune outcome, indicative of epigenetic or environmental influences on phenotypic expression; iv) innate immunity, particularly complement defects, as well as adaptive immunity, is complicit in the immunodeficiency-autoimmunity axis; v) features of certain of the disorders discussed at the meeting forced a reappraisal of what actually is meant by 'autoimmune disease'. It was concluded that genes that determine inherited immunodeficiencies, hitherto rather neglected by autoimmunologists, compel attention to consideration of molecular genetic anomalies critical for emergence of autoimmune disease in humans and animals.

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease in which an immune-mediated injury targets the small intrahepatic bile ducts. PBC is further characterized by highly specific serum antimitochondrial autoantibodies (AMA) and autoreactive T cells, a striking female predominance, a strong genetic susceptibility, and a plethora of candidate environmental factors to trigger the disease onset. For these reasons PBC appears ideal to represent the developments of the clonal selection theory over the past decades. First, a sufficiently potent autoimmunogenic stimulus in PBC would require the coexistence of numerous pre-existing conditions (mostly genetic, as recently illustrated by genome-wide association studies and animal models) to perpetuate the destruction of the biliary epithelium by the immune system via the persistence of forbidden clones. Second, the proposed modifications of mitochondrial autoantigens caused by infectious agents and/or xenobiotics well illustrate the possibility that peculiar changes in the antigen structure and flexibility may contribute to tolerance breakdown. Third, the unique apoptotic features demonstrated for cholangiocytes are the ideal setting for the development of mitochondrial autoantigen presentation to the immune system through macrophages and AMA thus turning the non traditional mitochondrial antigen into a traditional one. This article will review the current knowledge on PBC etiology and pathogenesis in light of the clonal selection theory developments.

Using a simple screening technique called regression of offspring on mid-parent (ROMP) to examine the role of IgG subclasses in affected and unaffected siblings of children and adolescents with autoimmune thyroid disease and their parents, both total-restricted and subclass-restricted autoantibodies to thyroglobulin (Tg) were assayed quantitatively for each of the IgG subclasses. There was a significant correlation of anti-Tg titer of probands with parental titers in thyrotoxicosis (TT), (R(2) = 0.569, p = 0.001), but not in chronic lymphocytic thyroiditis. The most striking correlation was in TT patients of African-American ancestry, (R(2) = 0.9863, p = 0.0007). Additional insight is provided by examining the contributions of the IgG subclasses individually, particularly those whose concentrations appear not to have direct influence on the total IgG titers. Thus, using small numbers of patients, and assaying the IgG subclass distributions, as well as any other immunoglobulin isotypes that are significantly altered in autoantibody assays, ROMP can be performed rapidly to ascertain which quantifiable parameters may be usefully extended to predict disease onset and progression.

New linguistic coinage can signify new practices and fresh perceptions in science: descriptors therefore are not trivial. Here, we consider the shifting valence of 'allergic' and 'autoimmune' in conceptions of experimental encephalomyelitis (EE). Ehrlich's dismissal of the relevance to disease of autoimmunity resulted in its 'long struggle for recognition' notwithstanding the convincing attribution in 1904 of the hemolysis of paroxysmal cold hemoglobinuria. Yet allergy did take hold because of its assumption that harmful effects could be ascribed to an extrinsic agent against which immune responses were supposed to be directed, in line with contemporary microbiological research. In 1885 the history of EE began with Pasteur's anti-rabies vaccine, dried virus-infected rabbit spinal cord, with use occasionally inducing a post-vaccinal encephalomyelitis (PVE). From 1933 to 1935, PVE was investigated by Rivers who reported that some monkeys immunized with normal rabbit CNS extracts developed an inflammatory demyelinating EE and anti-brain antibodies: no cause was attributed. In the 1940s Freund developed an adjuvant that greatly potentiated immunization and in 1947 this was applied to animals immunized for EE: induction was accelerated and the disease was called 'E allergic E', initiating the EAE acronym. As recorded, 'the study of autoimmune disease leapt from nothing in 1945 to a vigorous field in the 1950s'. Yet researchers sedulously retained allergic in the EAE acronym until the1980s, long after 'autoimmune' had become available to them. Eventually practitioners for whom autoimmunity had meaning influenced the transition to 'E autoimmune E' as the laboratory analogue of human autoimmune multiple sclerosis.

We briefly survey the concept of autoimmunity and nominate the range of autoimmune diseases that include multisystemic and organ-specific disorders, and cite prevalences of autoimmune diseases in males and females, in humans and in experimental animals. Most human autoimmune diseases such as systemic lupus erythematosus (SLE), multiple sclerosis (MS) and autoimmune thyroid disease, have an increased incidence and prevalence in females, but a few others such as autoimmune diabetes, the Guillain Barré syndrome (GBS) and psoriasis are increased in males. Animal models of autoimmunity show an equivalent sexual dimorphism. The possible reasons for the differing incidence and prevalence of autoimmune diseases in females and males engage our attention. Environmental exposures may differ for females and males. There are innate differences in the function of the female and male immune systems, and there is some evidence for differences between females and males in the ability of a target organ for autoimmunity to withstand damage. In seeking reasons for these differences, we review the role of sex hormones in immunity and include results of trials of hormone therapy in autoimmune diseases. The association of autoimmunity and pregnancy, a female-specific condition, is discussed, and the claimed effects of lymphoid cell microchimerism on provocation of autoimmunity are reviewed. Genetic predisposition is an important factor in autoimmune disease and we particularly focus on genes on the X and Y chromosomes, the role of X chromosome inactivation, and the interaction of the sex of the patient with other genetic factors. The possible role of epigenetic mechanisms, including environmental influences, is then surveyed. We assert that sex is a vital variable that must be considered in all immunological studies, as it should be at all levels of biological research.

Improvements in the treatment of primary biliary cirrhosis (PBC) may depend upon dissection of mechanisms that determine recruitment of mononuclear cells to intralobular bile ducts, including the role of the chemokine-adhesion molecule CX3CL1 (fractalkine). We submit that there are unique interactions between intrahepatic biliary epithelial cells (BECs), endothelial cells (ECs), liver sinusoidal endothelial cells (LSECs), and liver-infiltrating mononuclear cells (LMCs), and that such interactions will in part dictate the biliary-specific inflammatory response. To address this, we studied fresh explanted livers from pretransplantation patients with PBC and with inflammatory liver disease due to viral infection (disease controls) and biopsy material from patients with a discrete liver tumor (normal controls). Using this clinical material, we isolated and stimulated BECs, ECs, LSECs, and LMCs with a panel of Toll-like receptor ligands. We also studied the interactions of these cell populations with LMCs with respect to adhesion capability and production of tumor necrosis factor alpha (TNF-alpha). Finally, we used fresh biopsy samples to evaluate mononuclear cells around intrahepatic biliary ductules using monoclonal antibodies specific to CD68 or CD154, markers for monocytes/macrophages, and activated T cells, respectively.There are common properties of ECs, LSECs, and BECs, whether derived from PBC or viral hepatitis, but there are also significant differences, particularly in the potential in PBC for LMCs to adhere to ECs and BECs and to produce TNF-alpha; such properties were associated with augmented CX3CL1 production by BEC from PBC liver. The processes defined herein suggest potential novel biotherapies for biliary specific inflammation.

The concept that autoimmune diseases are characterized by shared (common) threads is well illustrated by their propensity to co-associate in a patient or direct relatives, as coexistences or overlaps. Recognized are two major autoimmune clusters, “thyrogastric” (mostly organ-specific) and “lupus-associated” (mostly multisystem). Additionally, some autoimmune diseases distribute within either cluster and a few appear not to associate. Also, within each cluster there are overlaps constituting virtually a distinct syndrome. These patterns of coexistence/overlaps depend predominantly on genetic determinants as judged by data accruing from numerous highly powered genome-wide association studies. Gene polymorphisms thus revealed include those that may determine tissue targeting particularly HLA alleles (and others), the (numerous) genes that influence orderly progression (or tolerogenesis) among immune responses from innate immunity to effector processes, genes that influence pathways of apoptosis, and genes that influence vulnerability of target organs to immune-mediated damage. A telling illustration is provided by immune-mediated colonic diseases wherein there is now a demonstrated capability to allocate the multiple risk genes to either Crohn's disease, ulcerative colitis, or both as inflammatory bowel disease. Precisely accurate clinical data-bases and serological reactivities, combined with deepening molecular genetic analyses, have much promise for a better understanding of autoimmunity.

A man, aged 34, was treated in 1954 for duodenal ulcer by antroduodenectomy followed by X-irradiation to the stomach in a dose of 2,000 rads. Over two decades, he developed several conditions attributable to the previous irradiation, including the physical appearances of premature ageing, shrinkage of the left kidney due to irradiation nephritis, immune deficiency, multiple myeloma of IgA type, and lastly, carcinoma of the stomach. The kidneys, especially the left, the bone marrow and stomach would have been in the field of X-irradiation. These effects of local X-irradiation are discussed in relation to the known effects of total body irradiation in causing decreased longevity in animals and inducing cancer in man.

SummaryOur laboratory has suggested that loss of tolerance to pyruvate dehydrogenase (PDC-E2) leads to an anti-mitochondrial antibody response and autoimmune cholangitis, similar to human primary biliary cirrhosis (PBC). We have suggested that this loss of tolerance can be induced either via chemical xenobiotic immunization or exposure to select bacteria. Our work has also highlighted the importance of genetic susceptibility. Using the non-obese diabetic (NOD) congenic strain 1101 (hereafter referred to as NOD.1101 mice), which has chromosome 3 regions from B6 introgressed onto a NOD background, we exposed animals to 2-octynoic acid (2OA) coupled to bovine serum albumin (BSA). 2OA has been demonstrated previously by a quantitative structural activity relationship to react as well as or better than lipoic acid to anti-mitochondrial antibodies. We demonstrate herein that NOD.1101 mice immunized with 2OA-BSA, but not with BSA alone, develop high titre anti-mitochondrial antibodies and histological features, including portal infiltrates enriched in CD8+ cells and liver granulomas, similar to human PBC. We believe this model will allow the rigorous dissection of early immunogenetic cause of biliary damage.

— In primary biliary cirrhosis (PBC), autoantibodies are produced to the M2 group of mitochondrial antigens, of which a major constituent is the E2 subunit of the pyruvate dehydrogenase complex (PDC). These antibodies, in addition to conventional reactivities with PDC, characteristically inhibit the catalytic function of PDC in vitro, as judged by a macroinhibition assay based on spectrophotometry. We describe a microinhibition assay adapted for microtitre plates and for an automated readout of results by an ELISA plate reader. We show that this microassay has a sensitivity similar to that of the macroassay. In a study of 83 sera, from PBC and other diseases, the inhibitory microassay proved specific for PBC. This automated inhibitory microassay for PBC sera could become a primary laboratory procedure for the diagnosis of PBC, particularly because it may identify antibodies to the actual autoepitope on the PDC-E2.

Aims To compare (i) the prevalence and incidence of chronic complications and (ii) cardiac and all-cause mortality in community-based patients with latent autoimmune diabetes in adults (LADA) with those in Type 2 diabetic patients without antibodies to glutamic acid decarboxylase (GAD). Methods Of the 1294 patients with clinically-defined Type 2 diabetes recruited to the longitudinal, observational Fremantle Diabetes Study between 1993 and 1996, 1255 (97%) had GAD antibodies measured at baseline. Complications were ascertained using standard criteria in patients returning for annual assessments until November 2001. Data on hospital admissions and mortality were available to the end of June 2006. Cox proportional hazards modelling was used to determine independent predictors of first occurrence of complications and cardiac and all-cause mortality. Results Forty-five (3.6%) subjects had LADA. Compared with the GAD antibody-negative patients, they had a similar prevalence and incidence of coronary heart ( P = 0.48 and 0.80, respectively) and cerebrovascular ( P = 0.64 and 0.29) disease and cardiac and all-cause mortality ( P = 0.62 and 0.81, respectively). There was also a similar prevalence and incidence of retinopathy ( P = 0.22 and 0.64, respectively) and neuropathy ( P = 0.25 and 0.95), but microalbuminuria was less frequent both at baseline and during follow-up in the LADA subgroup in unadjusted models ( P = 0.046) and after adjustment for other risk factors ( P = 0.014 and 0.013). Conclusions Except for a lower prevalence and incidence of nephropathy, LADA patients have a similar risk of complications and death to patients with clinically-diagnosed Type 2 diabetes without GAD antibodies. Cardiovascular risk factor management in LADA should, therefore, be as intensive as that for GAD antibody-negative patients.

Approximately 5% of patients with primary biliary cirrhosis (PBC) lack characteristic anti-mitochondrial antibodies (AMA). Yet clinically AMA+ and AMA- patients are similar. Using both AMA+ and AMA- patients, we quantitated the frequency of autoreactive T cells that respond to the major CD4 T-cell epitope, PDC-E2 163-176, using limiting dilution assays and quantitation of IFN-gamma, IL-10 and IL-4. Further, based on data that both PBC patients and healthy subjects have CD4+ T cells that recognize PDC-E2 163-176 but with differing costimulation requirements, assays were performed using two different antigen-presenting cell (APC) systems: either autologous peripheral blood mononuclear cells (PBMC) or HLA DR53 transfected mouse fibroblast cell lines (L-DR53). When costimulation-incompetent L-DR53 were used as APCs, the PDC-E2 CD4 T-cell frequency and capacity for IFN-gamma production were equivalent in both AMA+ and AMA- patients but the frequencies of such cells were significantly lower in normals, with IL-10 production similar in all three groups. Thus, in PBC there is 'universal' autoreactive CD4+ T-cell immune responsiveness to the critical autoantigen, PDC-E2. These observations emphasize that the mitochondrial autoreactivity in PBC is a multi-lineage response and hence, AMA-negative PBC may be an anachronism that refers only to sera autoantibodies.

The detection of serum autoantibodies to smooth muscle (SMA) on rodent gastric mucosa by indirect immunofluorescence (IIF) has long been an immunodiagnostic marker for autoimmune hepatitis type 1 (AIH-1). The reactive antigenic moieties are cytoskeletal proteins which include polymeric F-actin as judged by the staining of microfilaments of tissue by IIF. However, their specificity for actin in AIH-1 can be and usually is uncertain. Using an in vitro functional assay, we compared the effects of Fab fragments of immunoglobulin (IgG) prepared from SMA-positive plasma from two patients with the effects of Fabs from 10 healthy subjects. Fabs are incorporated into an assay where actin (the putative antigen) activates skeletal muscle heavy meromyosin (HMM) ATPase activity. The data from these functional assays provide new insights into the significance of anti-microfilament assays in the diagnosis, and perhaps also pathogenesis, of AIH-1.

Interleukin-2 (IL-2) receptor α knockout (IL-2Rα−/−) mice have a deficiency of CD25 and a corresponding functional defect in T regulatory cells (Tregs). These mice spontaneously develop portal inflammation with biliary ductular damage and colitis with features similar to human inflammatory bowel disease with T cell infiltrates in both the liver and colon. In humans, inflammatory bowel disease may be accompanied by primary sclerosing cholangitis (PSC), but seldom primary biliary cirrhosis (PBC). We hypothesized that the effector mechanism responsible for T cell infiltrates would differ for colon versus liver. To address this thesis, we developed three colonies of double-knockout mice including IL-2Rα−/− CD4−/−, IL-2Rα−/− CD8−/−, and IL-2Rα−/− T cell receptor (TCR)-β−/−. Tissue immunopathology, body weight, and serum levels of cytokines, immunoglobulins, and anti-mitochondrial antibodies (AMA) were assayed at 3 months of age. Relative to IL-2Rα−/− mice, IL-2Rα−/− CD4−/− mice had increased biliary ductular damage but reduced inflammation in the colon. In contrast, IL-2Rα−/− CD8−/− mice had increased colon inflammation but markedly attenuated biliary ductular damage. Both IL-2Rα−/− CD4−/− and IL-2Rα−/− CD8−/− mice demonstrated elevated serum levels of tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin-12p40 (IL-12p40), and interleukin-2 (IL-2) compared with C57BL/6J controls, but only IL-2Rα−/− CD8−/− mice had increased serum levels of immunoglobulin A (IgA), AMA and interleukin-17 (IL-17). Finally, and of importance, IL-2Rα−/− TCR-β−/− mice had abrogation of liver and colon pathological conditions and lacked AMA. In conclusion, on loss of Treg function in mice, CD8 T cells mediate biliary ductular damage whereas CD4 T cells mediate induction of colon-specific autoimmunity. (HEPATOLOGY 2009;49:133-140.)

The binding of radioiodinated basic protein of myelin ([125I]BPM) to a sheep lymphocyte cell pellet, sheep erythrocytes, and cell-free tubes used in the assay was investigated as a possible diagnostic procedure in multiple sclerosis. [125I]BPM apparently bound to 5 x 10(6) sheep lymphocytes incrementally with no plateau, up to 300 ng, and also to sheep erythrocytes; when cells were transferred to fresh tubes, over 90% of the radioactivity remained on the original tube, regardless of the tube surface. Various substances, including BPM and unrelated basic proteins, competitively inhibited the binding of basic protein of myelin to sheep cells and assay tubes. Binding was inhibited by sera from patients with multiple sclerosis, but equally so by normal sera. The large capacity of BPM to bind nonspecifically could limit its use in the above type of binding assay and would need to be allowed for in conventional radioimmunoassays.

Regulatory T (Treg) cells are important contributors to the maintenance of immune tolerance in the periphery, and deficiency of Tregs is associated with various immunopathic diseases. Murine models of autoimmune and autoinflammatory disorders have helped to elucidate how Tregs are involved in these diseases. A feature in common between human and mice that lack one or another of the key Treg subsets is the occurrence of mucosal inflammation. The relatively fragile mucosal surface represents a complex system that is normally well equipped to ward off harmful pathogens yet at the same time is inhibitory to destructive inflammatory responses to biologically needed (probiotic) microorganisms, or other common environmental antigens e.g. nutrients. We here discuss the importance of Tregs in maintaining tolerance at mucosal surfaces and the outcomes of deficiency of Treg function. The intestinal tract and its inflammatory diseases provide the "point of departure" for discussion, but similar considerations could apply to other mucosal linings exposed to the environment such as other members of the digestive system. However, the lungs, bile ducts, urogenital tract and other mucosal surfaces are susceptible to poorly understood inflammatory states that possibly depend on dysfunction of Treg cells. Finally there are now potential therapies predicated on reconstitution of effective function of Treg cells.

Knowledge on autoimmunity is examined from the launch of clonal selection theory 1957-1959. Crucial elements then were 'forbidden clones' of immunocytes as agents of tissue damage, somatic mutations that generated such clones and 'homeostatic mechanisms' that controlled them. The understanding of autoimmunity over the succeeding 50 years has expanded immensely, and many more diseases now come under this rubric. Examined here are current problems of definition including 'adaptive' and 'innate' types of autoimmunity, estimations of population burdens of autoimmune diseases, the nature of autoepitopes in the context of the diabetes autoantigen GAD65, and the complexities of immune tolerance and the genetic influences thereon, leading to the nomination of multiple 'tolerance/autoimmunity' genes as critical components of pathogenesis. Burnet's concept of mutagenesis as a basic feature of various pathologies including autoimmunity is given a contemporary focus, his views on deletional tolerance have been well vindicated, his 'forbidden clones' remain as unphysiological as before albeit phenotypically resembling normal lymphocytes, and his 'homeostatic mechanisms' can be now interpreted in terms of immunoregulatory networks.

Autoimmune liver diseases (AILD) including autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC) have attracted much attention since their discovery 50 years ago, but there remain items of unfinished business. These relate to disease susceptibility including genetic influences (HLA and non-HLA genes, genes associated with female predisposition, and others) and environmental influences (infections, chemicals, xenobiotics and medications). Also needed is better characterization of autoantigenic molecules, particularly the anti-F-actin specificity characteristic of AIH, shown here to have functional effects in vitro. Deeper analysis of T-lymphocyte function in AILD should reveal relative contributions of eachof the multiple subsets of T cells now being defined in studies on laboratory animals, CD4(+), CD8(+), Th1, Th2, Th17, memory subsets and regulatory subsets. Diagnostic immunology providers now offer high-performance assay formats that call for systematic clinical assessments to achieve standardization, calibration and optimal information.

CCR5 is a chemokine receptor that mediates entry of human immunodeficiency virus-1 (HIV-1). Two monoclonal antibodies (mAbs) that block HIV-1 entry, 3A9 and 5C7, were used to select peptide mimotopes of sequences on CCR5 from phage displayed peptide libraries. The selected mimotofpes comprised motifs at the N-terminus and on the first and third extracellular loops (ECL1 and ECL3) of CCR5. Amino acids in these motifs were exchanged for alanines by site-directed mutagenesis (sdm) in the cDNA for human CCR5. Ensuing effects on antibody binding to CCR5, cellular entry of HIV-1 and chemokine-induced signalling were analysed by transfection of mutant cDNAs into HEK293.CD4 cells. For both mAbs, fluorescence-activated cell sorting analysis was used to define overlapping conformational epitopes on CCR5 at the N-terminus, on ECL1 and ECL3. Mutation of the N-terminal motif 10YD11 prevented HIV-1 entry into transfected cells as judged by single round infection assays with R5 and R5X4 HIV-1 isolates, as did mutation of the motif 96FG97 in ECL1, whereas mutation of the motif 274RLD276 in ECL3 had only a minor effect. None of the motifs in CCR5 relevant to HIV-1 entry disrupted chemokine-induced signalling. Thus, peptide mimotopes of conformational contact sites of CCR5 with the paratope of mAbs 3A9 and 5C7 represent sites on CCR5 that are essential for HIV-1 entry. Structural knowledge of these mimotopes could help elucidate the nature of the interaction between CCR5 and HIV-1, and thus the derivation of specific inhibitors of entry of HIV-1 into susceptible cells without interference with chemokine signalling.

Background Among numerous studies in the literature regarding prognostic factors that might determine outcome in functional endoscopic sinus surgery (FESS), very few have dealt with assessment of their predictive potential regarding outcome, and none have tried to find out the extent of such a prediction. We have developed a composite model to find out the predictive values of various prognostic factors, using an outcome measure based on the need for postoperative medical intervention. Methods One hundred nine patients were recruited for FESS and followed up at regular intervals for 5 years. Ten prognostic factors were identified and were correlated with prospectively recorded outcome measures. A discriminant analysis using SPSS software was performed to identify the prognostic factors that could “predict” the outcome. Results CT scan scores and polyp scores were the strongest predictors. Using our model, the probability to predict correctly the need for postoperative systemic medication was found to be 81.7%. Conclusion We have generated a model to predict the outcome of FESS, with a 5-year follow-up. CT scan scores and polyp scores were found to be the strongest predictors of the need for postoperative systemic medication.

From the immunology standpoint, the liver is a peculiar organ for several reasons that range from its anatomical location to its cytoarchitecture and its variety of specific functions. Receiving blood directly from the digestive system, the liver is the crossroad at which the majority of antigens enter the organism. Hence, the milieu of the liver must provide a finely tuned balance between generating tolerance to self as well as to nonpathogenic molecules and microorganisms and producing an appropriate immune response to pathogens. Knowledge of the mechanisms that effectively maintain this balance is critical to the understanding of the pathogenesis of chronic inflammatory liver diseases, infectious, autoimmune, and others. Several theories have been proposed to explain what causes one or another response to take precedence. Although no definitive answer is yet available, the critical elements include, for tolerance, the particular cytoarchitectural features and the intrahepatic existence of antigen-presenting cells and, for immunity, inflammatory expressions including type 1 cytokines and chemokines, notably CCR5. Herein we review the available data on immune responses in the liver with particular emphasis on the unique structural and functional features of this "lymphoid" organ.

Autoantibodies to the 65 kDa isoform of glutamate decarboxylase (GAD65) are associated with type I diabetes and recognise highly conformational epitope(s) that remain to be defined. The human recombinant Fab from mAb b96.11 inhibits binding of most GAD65 antibody positive sera from patients and its epitope has previously been localized to the middle region of GAD65. Recent studies indicate that b96.11 antibody specificity predicts the risk of developing type 1 diabetes in prediabetic individuals. We describe the use homology modelling, protein–protein docking simulations and biopanning of random peptide phage displayed libraries with b96.11 to predict contact amino acids on the interface of GAD65/Fab b96.11 complex. Further analysis by in vitro mutagenesis of GAD65 followed by radioimmunoprecipitation refined the amino acids contributing to the b96.11 epitope. Our studies show an interface characterized by a protruding antibody-combining site centered on the long heavy chain CDR3 loop of Fab b96.11 establishing interactions with the critical residue Phe 344 in the core of the epitope on GAD65, surrounded by charged sites within 375 RK 376 and 305 DER 307 . The epitope requires residues from both middle and the C-terminal domains, and is the first precise definition of an epitope on GAD65. The nature of the b96.11 epitope leads to considerations of potential structural variations for differences in antigenicity between the isoforms GAD65 and GAD67. The study shows the utility of using a combination of in silico techniques and experimental data for molecular characterization and localization of conformational epitopes for which crystal structures are lacking.

Autoantibodies to the diabetes autoantigen, the 65 kDa isoform of glutamic acid decarboxylase (GAD65), react with conformational epitopes defined according to linear sequences but not according to structural information, or contact sites with the antibody paratope. To ascertain such information for an exemplary human monoclonal antibody (mAb) to GAD65, b78, we combined antibody screening of phage-displayed peptide libraries, alanine mutagenesis of selected motifs, homology modelling of the PLP and C-terminal regions of GAD65, and molecular dynamics to examine for structural effects of mutagenesis. By phage display, mAb b78 selected phagotopes containing acidic residues (D, E), hydrophobic residues (Y, F or W) and LRS that localised to a possible surface-exposed conformational epitope on the combined homology model. Alanine mutants of GAD65 based on deduced contact residues were examined for binding with b78 and control sera. Mutation of 524 SRL 526 , 572 DF 573 and 498 KPQ 500 reduced reactivity of b78 with mutant GAD65 > 50%. Molecular dynamics indicated that mutation of 498 KPQ 500 caused structural changes that could account for effects of this mutation. Thus phage display in combination with molecular modelling identified contact residues within a highly conformational epitope for mAb b78 in the C-terminus of GAD65. These techniques should have broad applicability to definition of epitope structure.

Primary biliary cirrhosis (PBC) is an autoimmune disease with a strong genetic component characterized by biliary ductular inflammation with eventual liver cirrhosis. The serologic hallmark of PBC is antimitochondrial antibodies that react with the pyruvate dehydrogenase complex, targeting the inner lipoyl domain of the E2 subunit (anti–PDC-E2). Herein we demonstrate that NOD.c3c4 mice congenically derived from the nonobese diabetic strain develop an autoimmune biliary disease (ABD) that models human PBC. NOD.c3c4 (at 9–10 wk, before significant biliary pathology) develop antibodies to PDC-E2 that are specific for the inner lipoyl domain. Affected areas of biliary epithelium are infiltrated with CD3+, CD4+, and CD8+ T cells, and treatment of NOD.c3c4 mice with monoclonal antibody to CD3 protects from ABD. Furthermore, NOD.c3c4-scid mice develop disease after adoptive transfer of splenocytes or CD4+ T cells, demonstrating a central role for T cells in pathogenesis. Histological analysis reveals destructive cholangitis, granuloma formation, and eosinophilic infiltration as seen in PBC, although, unlike PBC, the extrahepatic biliary ducts are also affected. Using a congenic mapping approach, we define the first ABD (Abd) locus, Abd1. These results identify the NOD.c3c4 mouse as the first spontaneous mouse model of PBC.

The E2 subunit of the mitochondrial multienzyme pyruvate dehydrogenase complex (PDC–E2) is the major autoantigen in the liver disease, primary biliary cirrhosis (PBC). An epitope region which has been localized to amino acids 91–227 is believed to include the residue K173 to which is attached the lipoyl cofactor. We investigated structural features of this epitope region by screening random peptide phage-displayed libraries and identified prevalent phagotopes that contained likely contact amino acids in separate regions of the linear sequence, H132M133, and F178, V180. These were confirmed by site-directed alanine mutagenesis singly or in combination of the HM and FV residues in wild-type (wt) PDC–E2, and by immunization of rabbits with phage that expressed peptides MHLNTPP or FVLPWRI. The lipoyl lysine K173 also was mutated. Reactivities of mutants and wild-type (wt) PDC–E2, compared by ELISA using 12 PBC sera, showed decremental reactivity of mutant versus wt PDC–E2 (normalized to 100%): wt PDC–E2 (100%) ≫ PDC–E2F178A,V180A (mean ± S.D., 59 ± 17%) > PDC–E2M133A (50 ± 13%) > PDC–E2H132A (36 ± 13%) > PDC–E2H132A,M133A (28 ± 8%) > PDC–E2H132A,M133A,F178V,M180A (18 ± 13%). Notably PDC–E2K173A retained full reactivity (93 ± 21%). Rabbits immunized with phage peptides generated antibodies reactive with entire PDC–E2. Our data convincingly validate phage library technology for defining spatially disparate contact residues for conformational epitopes. Ensuing data could be generally applicable to search for occult extrinsic agents as initiators of autoimmunity.

Molecular mimicry between the 65-kDa isoform of glutamic acid decarboxylase (GAD65) and the protein 2C (P2C) of Coxsackie B4 virus (CBV) may initiate human type 1 diabetes. GAD65 contains a motif that has a 6-amino acid identity with CBV-P2C (PEVKEK), whereas the weakly autoantigenic isoform, GAD67, contains PEVKTK. A human-derived monoclonal antibody (mAb) MICA3 reacts with a surface loop of GAD65 that includes PEVKEK, and mutagenic deletion of this loop was shown to reduce reactivity of GAD with the mAb by 70%. To establish that the PEVKEK motif on GAD65 contains a major epitope for diabetes sera and to identify the amino acids involved, mutants of nucleotides of GAD65 and GAD67 at sites in the PEVKEK motif were created and the expressed proteins used for radioimmunoprecipitation (RIP) tests with sera from patients with type 1 diabetes. A potent mouse mAb (GAD6) to GAD65, and a rabbit polyclonal antibody (AB108) to GAD67, were used to standardize the reactivity of the diabetes sera with the mutant molecules. Of 45 type 1 diabetes sera tested, 30 (67%) had an 80% or greater reduction of reactivity to GAD65(delta258-270) vs. intact GAD65. Various single-surface amino acids in the PEVKEK epitope region of GAD65 were mutated, but most molecules carrying these mutations reacted similarly to the parent molecule. However after point mutation of the equivalent motif of GAD67 (PEVKTK to PEVKEK), there was an increase in the reactivity of 12 of 49 (24%) type 1 diabetes sera tested; 7 of 8 sera reactive with GAD67 showed increased reactivity with GAD67(T273E), and 5 previously negative sera gained reactivity with GAD67(T273E). Thus, the PEVKEK motif on GAD65 contributes to serologic reactivity of type 1 diabetes sera. This favors the hypothesis that CBV infection causes type 1 diabetes by the process of viral mimicry with cross-reactivity to a critical epitope of GAD65.

Recently, we identified a child born with a genetic deficiency of IL-2 receptor alpha (IL-2Ralpha, CD25) expression who had several clinical manifestations of primary biliary cirrhosis (PBC). In addition, there has been suggestive evidence in both patients with PBC and their first-degree relatives that a deficiency of regulatory T cells (Tregs) is an integral component for susceptibility to PBC. Based on these observations, we generated IL-2Ralpha/CD25 deficient (IL-2Ralpha(-/-)) mice and wild-type littermate controls and followed them longitudinally for the natural history of liver immunopathology and appearance of antimitochondrial antibodies (AMAs). The analyses included immunohistochemical staining of liver and portal tract infiltrates as well as FACS profiles of lymphoid subpopulations in liver and spleen. In addition, serum cytokine profiles were quantitated. Importantly, IL-2Ralpha(-/-), but not littermate controls, develop portal inflammation and biliary ductular damage similar to human patients with PBC. CD4(+) and CD8(+) T cells predominate among portal cell infiltrates and sera reflect a Th1 cytokine bias with increased levels of IFN-gamma, TNF-alpha, IL-2 and IL-12p40. Of importance is the finding that the IL-2Ralpha(-/-) mice not only develop significantly increased serum levels of IgG and IgA, but they also develop AMAs with specificity for PDC-E2, which maps to the inner lipoyl domain of the autoantigen, all characteristics which are hallmarks of human PBC. In conclusion, the IL-2Ralpha(-/-) mice should facilitate studies of the early events in PBC and especially the tantalizing connection between Treg deficiency and autoimmunity specifically directed to mitochondrially located PDC-E2 and subsequent biliary ductular cell damage.

Acquisition by mammals of an adaptive immune system was an evolutionary leap, but occurred at the cost of autoimmunity. The necessity for self-recognition was appreciated in 1900, but autoimmune disease did not become a clinical reality until the 1950s-still the perimeters are indistinct. Autoimmune responses recapitulate the complex events of normal immune responses but cannot shut down. Immune tolerance is established during repertoire development centrally in thymus or bone marrow by deletion of self-reactive immunocytes, and is supplemented peripherally by regulatory T cells (Tregs). A startling discovery is the autoimmune regulator AIRE gene that enables expression of organ-specific autoantigens for intrathymic deletional tolerance of T cells. The origins, activities, and markers of Tregs are under intensive investigation. Of genes implicated in autoimmune, only few are characterized; contributions of environment are similarly uncertain. Time-latency considerations implicate stochastic factors or chance in the etiopathogenesis of autoimmunity, whether they are somatic mutations or successive random gene-environment interactions. Solutions to etiopathogenesis require novel experimental models and finely designed gene-environment studies on human populations. Perhaps immunomodulatory therapies will effect cures before causes are fully understood.

The HLA DQB1*0302 allele on DR4 haplotypes is a well-known marker for T1D susceptibility. The contribution of this DQ molecule to overall disease susceptibility may be genotype dependent. On Asian DR4 haplotypes, not only DQB1*0302, but also DQB1*0401 confer susceptibility to T1D, which is mediated by DRB1*0405 on the same haplotype. To analyze further the association of DQB1*0302 and DQB1*0401 haplotypes in Asian patients with T1D, we investigated HLA DR and DQ alleles with the presence or the persistence of GAD, ICA, and IA-2 autoantibodies in 121 Korean T1D patients. In Korean patients, there was an association of IA-2 autoantibodies with DR4 as well as DR3, but not with DQA1*0301-DQB1*0302. GAD autoantibodies were not associated with the DR3-DQB1*0201, DQA1*0301-DQB1*0302, or DR4. In this low-risk population, autoantibodies to islet-cell antigens were associated with different HLA molecules. In different populations, the immune response to different beta cell autoantigens is mediated via varying HLA class II molecules from different loci. Design of the antigen-specific immunointervention trials should take into account such HLA DR and DQ association.

Humanism includes, among its many contexts, the ideal of the universal perfection of health. Procedures for alleviation of disease existed through all epochs of human history, but efficacy was mostly lacking. The prototypic humanism of the Renaissance (ad 1300-1600) scarcely involved the medical -sciences other than human anatomy. The Enlightenment of the seventeenth century included discovery of the circulation of the blood, and applications of microscopy. Discoveries relevant to medical practice began in the nineteenth century, ushered in by vaccination and the germ theory of disease. This 200-year period saw a transformation of human health according to the surrogate marker of increased life--expectancy. This has been variously attributed to: (i) increased prosperity following the industrial revolution, (ii) efforts of humanistic social and public health reformers and, more recently, (iii) advances in medical science. Yet the beneficiaries remain a minority of the world's population. The nexus between poverty, illness and low life-expectancy between and within nations is the major challenge for the future. Contemporary science is providing ever-expanding knowledge on means to achieve the goal of perfection of human health, but the need for humanism is as great as at any previous age. Fortunately, however, the targets are more clearly visible than during the periods of poverty, plagues and pestilence of the past.

Aims The autoantigen SOX-13 of the SRY-related high mobility group box is a low-frequency reactant in sera from patients with Type 1 diabetes. We further investigated the potential diagnostic role of anti-SOX-13, and in particular its ability to distinguish Type 1 from Type 2 diabetes, in two large, well-characterized cohorts. Methods SOX-13 autoantibody status was ascertained using a radioimmunoprecipitation assay in (i) a random sample of 546 participants in an Australian community-based study (the Fremantle Diabetes Study; FDS) of whom 119 had Type 1 and 427 Type 2 diabetes, and (ii) a sample of 333 subjects with Type 2 diabetes from the United Kingdom Prospective Diabetes Study (UKPDS) stratified by age, anti-glutamic acid decarboxylase (GAD) and islet cell antibody (ICA) status, and requirement for insulin therapy within 6 years of diagnosis. Results The frequencies of anti-SOX-13 in the FDS subjects were 16.0% and 14.8% for Type 1 and Type 2 patients, respectively, and levels were similar. In the UKPDS subjects, the frequency was 4.5%. In a logistic regression model involving demographic, anthropometric and metabolic variables, only diabetes duration was significantly associated with anti-SOX-13 positivity, especially for duration > 5 years (P