Autoimmune Diagnostics 9783110228656, 9783110228649

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Autoimmune diagnostics Edited by Harald Renz

Autoimmune diagnostics Edited by Harald Renz

DE GRUYTER

Editor Prof. Dr. med. Harald Renz Institute of Laboratory Medicine Pathobiochemistry and Molecular Diagnostics Philipps University Marburg Baldingerstrasse D-35033 Marburg, Germany [email protected] This book contains 36 figs. and 27 tabs.

ISBN 978-3-11-022864-9 e-ISBN 978-3-11-022865-6 Library of Congress Cataloging-in-Publication Data A CIP catalog record for this book has been applied for at the Library of Congress Bibliographic information published by the Deutsche Nationalbibliothek The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available in the Internet at http://dnb.dnb.de. © 2012 Walter de Gruyter GmbH & Co. KG, Berlin/Boston Typesetting: Apex CoVantage, LLC Printing: Hubert & Co. GmbH & Co. KG, Go¨ttingen Cover image: Institut fu¨r Klinische Chemie des Universita¨tsklinikums Ko¨ln ⬁ Printed on acid-free paper s Printed in Germany www.degruyter.com

Contents

Preface ..................................................................................................................... ix Author Index ............................................................................................................ xi 1 General aspects of autoimmune diagnostics.......................................................... 1 1.1 General aspects and clinical features .............................................................. 1 1.2 Basis ............................................................................................................... 1 1.3 Scope of tests.................................................................................................. 3 1.4 Therapy .......................................................................................................... 4 1.5 Timing of investigations .................................................................................. 5 1.6 Storage and dispatch....................................................................................... 5 Hanns-Wolf Baenkler 2 Rheumatoid arthritis.............................................................................................. 7 2.1 General aspects and clinical features .............................................................. 7 2.2 Diagnostic workflow....................................................................................... 9 Rudolf Mierau and Ekkehard Genth 3 Systemic lupus erythematosus ............................................................................. 41 3.1 General aspects and clinical features ............................................................ 41 3.2 Diagnostic workflow..................................................................................... 43 3.3 Properties and diagnostic roles of SLE typical autoantibodies........................ 45 3.4 Prognostic value of autoantibodies................................................................ 50 Rudolf Mierau and Ekkehard Genth 4 Consensus protocol for the detection of autoantibodies by indirect immunofluorescence technology on HEp-2 cells................................................. 53 4.1 General aspects and clinical features ............................................................ 53 4.2 Reagents and test preparation ....................................................................... 53 4.3 Evaluation and interpretation ........................................................................ 57 Philipp von Landenberg 5 The antiphospholipid syndrome........................................................................... 63 5.1 General aspects and clinical features ............................................................ 63 5.2 Diagnostic measurements ............................................................................. 63 5.3 What tests should be used ............................................................................ 64 5.4 Test systems .................................................................................................. 64 5.5 The Lupus anticoagulant ............................................................................... 65 5.6 Recommendations ........................................................................................ 65 Philipp von Landenberg

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6 Inflammatory myopathies (polymyositis, dermatomyositis) ............................... 69 6.1 General aspects and clinical features .......................................................... 69 6.2 Diagnostic workflow................................................................................... 71 6.3 Properties and diagnostic roles of myositis-associated autoantibodies ............ 73 6.4 Prognostic value of autoantibodies.............................................................. 75 Ekkehard Genth and Rudolf Mierau 7 Systemic Sclerosis.............................................................................................. 77 7.1 General aspects and clinical features .......................................................... 77 7.2 Diagnostic workflow................................................................................... 78 7.3 Properties and diagnostic roles of SSc-typical autoantibodies...................... 80 7.4 Prognostic value of autoantibodies.............................................................. 83 Ekkehard Genth and Rudolf Mierau 8 Autoimmune blistering disorders ....................................................................... 89 8.1 General aspects and clinical features .......................................................... 89 8.2 Classification of autoimmune blistering dermatoses .................................... 89 8.3 Clinical characteristics ................................................................................ 89 8.4 Histopathology............................................................................................ 93 8.5 Immunofluorescence studies ....................................................................... 93 8.6 Immunoserologic analysis using recombinant or extractable autoantigens ............................................................................................... 94 8.7 Diagnostic pathways to autoimmune blistering disorders ............................ 97 Ru¨diger Eming and Michael Hertl 9 Autoimmune liver disease................................................................................ 103 9.1 General aspects and clinical features ........................................................ 103 9.2 Primary sclerosing cholangitis................................................................... 107 9.3 Primary biliary cirrhosis ............................................................................ 111 Stefan Lu¨th and Ansgar W. Lohse 10 Autoimmunity in diabetes mellitus .................................................................. 119 10.1 General aspects and clinical features ...................................................... 119 10.2 How does type 1 diabetes develop – the autoimmune pathogenesis .........119 10.3 Islet autoantibodies ................................................................................. 123 10.4 Type 1 diabetes risk screening ................................................................ 127 Peter Achenbach and Werner A. Scherbaum 11 Autoimmune thyroid diseases.......................................................................... 139 11.1 General aspects and clinical features ...................................................... 139 11.2 Etiology................................................................................................... 139 11.3 Clinical features ...................................................................................... 139 11.4 Workflow for the diagnosis of autoimmune thyroid disease .................... 140 Matthias Schott, Werner A. Scherbaum and Jochen Seissler

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12 Autoimmune primary adrenal insufficiency................................................... 157 12.1 General aspects and clinical features .................................................... 157 12.2 Etiology................................................................................................. 157 12.3 Clinical features .................................................................................... 158 12.4 Workflow for the diagnosis of autoimmune AD .................................... 158 12.5 Associated diseases ............................................................................... 160 Matthias Schott and Jochen Seissler 13 Inflammatory bowel disease .......................................................................... 163 13.1 General aspects and clinical features .................................................... 163 13.2 Serologic markers in IBD ...................................................................... 163 13.3 Diagnostic value of serologic markers................................................... 165 13.4 Serologic markers and clinical phenotypes ........................................... 166 13.5 Serologic markers in disease monitoring ............................................... 166 13.6 Therapeutic value of serologic markers................................................. 166 13.7 Subclinical value of serological markers ............................................... 167 Thomas Griga 14 Celiac disease ................................................................................................ 171 14.1 General aspects and clinical features .................................................... 171 14.2 Diagnostic workflow............................................................................. 171 Thomas Griga 15 Autoimmune polyendocrine syndromes......................................................... 177 15.1 General aspects and clinical features.................................................. 177 15.2 Autoimmune polyendocrine syndrome type 1 (APS-1) ........................ 177 15.3 Autoimmune polyendocrine syndrome type 2 (APS-2) ........................ 179 15.4 Clinical features.................................................................................. 179 15.5 Workflow for the diagnosis of APS ..................................................... 181 15.6 Gastrointestinal diseases ..................................................................... 184 15.7 Autoimmune hepatitis......................................................................... 184 15.8 Autoimmune hypoparathyroidism ....................................................... 185 15.9 Skin .................................................................................................... 185 15.10 Rare autoimmune manifestations of APS-1.......................................... 185 15.11 Prediction of subclinical autoimmune diseases ................................... 185 Matthias Schott and Jochen Seissler Index ..................................................................................................................... 191

Preface

In the mid-1990s the German Society of Clinical Chemistry and Laboratory Medicine (DGKL) founded the Autoimmune Diagnostics Working Group. It was initiated because the Society had become aware of the lack of standardized procedures in diagnostic laboratories specializing in autoimmune diseases. Standardization is of great importance because we note a tremendous increase, over the past 50 years, in the prevalence and incidence of virtually all chronic inflammatory conditions. This trend is largely due to a changing environment, particularly within westernized and industrialized societies, which has a strong impact on the maturation and development of normal immune responses. This situation has called for new strategies designed to diagnose patients as early as possible and to determine the causes of these increasingly common autoimmune diseases. Over the last few decades, a large number of newly developed diagnostic tests has been developed and has reached now clinical evaluation. These tests include genetic tests to assess individual risks for disease development, prognostic markers, and biomarkers for stratified (personalized) therapies. From the very beginning the Autoimmune Diagnostics Working Group was set up as an interdisciplinary enterprise in order to bring experts from the clinics together with experts from the diagnostic laboratory. In addition to a very strong core group of individuals, the working group was enlarged by key investigators bringing additional core expertise to the group. It was the group‘s aim to discuss and develop a diagnostic algorithm for all major autoimmune conditions prevalent in Europe. These algorithms start with the clinical presentation of patients and followed a stepwise approach for initial diagnosis and disease classification. Where appropriate, additional information was added regarding the prognostic value and therapy stratifying value of disease markers. The most difficult part was always to design a flowchart comprising all relevant information for diagnostic workflow. Such a template for the diagnostic chart was originally developed for rheumatoid arthritis and systemic lupus erythematosus and subsequently, where possible, adapted for other conditions. The working group, which was together for more than 15 years, recently merged with the newly founded section of immune diagnostics within the DGKL. This book is, therefore, designed not only to fill a gap within the diagnostic repertoire but also to mark the formal end of this years-long activity. As the chairman of this working group for more than 10 years, I would like to thank all of its members for their enthusiasm and positive spirit, which were evident in the more than 25 meetings we held throughout this period. I am sure that this activity and the book, as one result of this, will represent a starting point for future work in this very important area of laboratory medicine. Thanks to all members of our working group and the individual contributors and the continuous support and enthusiasm of our publisher, we are now able to present this new and comprehensive volume on autoimmune diagnostics. Harald Renz Marburg, 2012

Author Index

Dr. med. Peter Achenbach Forschergruppe Diabetes der Technischen Universita¨t Mu¨nchen Ko¨lner Platz 1 80804 Mu¨nchen e-mail: [email protected] Prof. Dr. Hanns-Wolf Ba¨nkler Friedrich-Alexander-University ErlangenNuremberg Department of Medicine 3 – Rheumatology and Immunology Krankenhausstrasse 12 D-91054 Erlangen e-mail: [email protected] Dr. med. Ru¨diger Eming Department of Dermatology and Allergology Philipps University Marburg Deutschhausstrasse 9 D-35037 Marburg e-mail: [email protected] Prof. Dr. med. Ekkehard Genth Hospital of Rheumatology Burtscheider Markt 24 D-52066 Aachen e-mail: [email protected]

Prof. Dr. med. Philipp von Landenberg Institut fu¨r Labormedizin ((Adresse in ¨ bersetzung folgt)) englischer U Solothurner Spita¨ler AG Kantonsspital Olten Basler Strasse 150 CH-4600 Olten e-mail: [email protected] Prof. Dr. med. Ansgar W. Lohse Department of Medicine I University Medical Center HamburgEppendorf Martinistrasse 52 D-24046 Hamburg e-mail: [email protected] PD Dr. med. Stefan Lu¨th Department of Medicine I University Medical Center HamburgEppendorf Martinistrasse 52 D-24046 Hamburg e-mail: [email protected] Dr. rer. nat. Rudolf Mierau Hospital of Rheumatology Burtscheider Markt 24 D 52066 Aachen e-mail: [email protected]

Prof. Dr. med. Thomas Griga Department of Internal Medicine Knappschaftskrankenhaus Dortmund Wieckesweg 27 D-44309 Dortmund e-mail: [email protected]

Prof. Dr. med. Harald Renz Institute of Laboratory Medicine, Pathobiochemistry, Molecular Diagnostics Baldingerstrasse D-35033 Marburg e-mail: [email protected]

Prof. Dr. med. Michael Hertl Department of Dermatology and Allergology University of Marburg Deutschhausstr.9 D-35037 Marburg e-mail: [email protected]

Prof. Dr. med. Werner Scherbaum University Hospital Du¨sseldorf Department of Endocrinology, Diabetes and Rheumatology Moorenstrasse 5 D-40225 Du¨sseldorf e-mail: Scherbaum@ddfi.uni-duesseldorf.de

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Author Index

Prof. Dr. med. Matthias Schott Endocrine Cancer Center Department of Endocrinology, Diabetes, and Rheumatology University Hospital Moorenstrasse 5 D-40225 Duesseldorf, Germany E-mail: [email protected]

Prof. Dr. med. Jochen Seißler Diabetes Center Medical Clinic Innenstadt Ludwig Maximilians University Munich Ziemssenstrasse 1 80336 Munich, Germany e-mail: [email protected]

1 General aspects of autoimmune diagnostics Hanns-Wolf Baenkler

1.1 General aspects and clinical features This chapter addresses the general facts, rules, and procedures that apply to diagnostic methods for various autoimmune diseases. Thus redundant information can be omitted in the following chapters, which focus on the details of autoimmune diagnostics in specific diseases. Here are discussed the basics of cellular and molecular mechanisms in lymphocytes and antibodies, recruitment, immune genetics, and concomitant systems such as complement system (uTab. 1.1). However, the main focus of this chapter is background information on the detection of autoantibodies by serologic methods.

1.2 Basis Autoimmune diseases and allergy derive from immune reactions that balance defense and self-tolerance. Whereas host defenses attack pathogens, allergies and autoimmune diseases are based on immune reactions against harmless antigens (allergens). Reactions may originate either from outside of the body (allergy) or from within it (autoimmune disease). Given the close relation, general approaches in the diagnostic procedures for both pathologic states are identical (1). The first evidence of a disease usually comes from the patient’s symptoms. The diagnostic approach aiming to demonstrate the pathologic reaction is highly dependent on the symptomatology. Since every chemical structure may become an antigen, there is a wide range of autoimmune diseases. Symptoms observed in patients with these diseases emerge from the contact of immune cells or their products with the antigen and depend on the type of reaction. Local disease manifestations are attached to a structure representing the antigen of an organ, a cellular population, or a soluble factor. Autoimmune diseases become systemic when the immune reaction is directed against common antigens or when immune complexes are disseminated throughout the body. The type of pathologic immune reaction determines symptoms and manifestations as well as the means for diagnosing the pathologic condition. Autoimmune diagnostics are based on chemical or physical tests, histology, and detection of specific immunologic activity. In this context different mechanisms of immune reactions govern the choice of applied techniques designed to measure cellular or soluble factors (2). The detection of autoantibodies before the autoimmune disorder becomes manifest is rare. Casual findings in healthy individuals indicate that it takes time to initiate damage and evoke symptoms. By contrast, in some diseases, no autoimmune phenomena are found on diagnostic evaluation, although inflammation and histological findings suggest an autoimmune disease. In some cases the beneficial effects of immunosuppressive

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1 General aspects of autoimmune diagnostics

Tab. 1.1: Immunologic approach to diagnostics Specific parameters Functional assay

Lymphocyte activation Autoantibody measurement

Immunologic imaging

Cytology/differentiation Histology/location

Immunogenetics

Compatability/probability

Nonspecific parameters Cooperative systems

Complement (components; total activity)

Interactive systems

Cytokines

drugs affirm this suspicion. Thus autoimmune disease cannot always be detected by routine diagnostic methods. Progress in methods and techniques optimizes diagnostics and helps to differentiate and interprete the test results. Biological and technical achievements steadily improve qualitative and quantitative results in diagnostics. Thus a vision seems to become reality: it becomes possible to establish a diagnosis of an autoimmune disease by new tests that provide substantial information beyond anamnesis and allow exploration in the early stages of the disease. This can provide substantial data regarding the future course of the disease and the patient‘s probable response to therapy. In this context it has to be mentioned that any comparison of results must respect the used methods and techniques, especially when tests are conducted in different laboratories. Autoantibodies are common targets in routine diagnostics and are therefore of great importance. The storage and the dispatch of probes for antibody diagnostics are relatively simple procedures and do not require high-tech facilities. Thus specific probes can be compared between different laboratories and different methods. This meets the need to control the diagnostic procedures and makes it possible to, for example, follow the spontaneous course of a disease or the response to treatment. Antibodies are heterogenous substances that deserve special considerations in the handling and applications of tests. Their heterogeneity results from their synthesis in several clones and from natural changes due to hypermutation. In a single clone, there is a switch affecting the isotype of the heavy chain but also a conformational change of the light chain. This process occurs between the initiated and the settled synthesis within a fully differentiated plasma cell for more effective antibody binding. It means a permanent increase of affinity, causing rising titers. This phenomenon happens at the molecular level after the first contact with the antigen; by contrast, stability prevails later on. Only the formation of agglutinins and the “classical” rheumatoid factor does not undergo the isotype switch. They are continuously of the IgM-type. This is important for the application of the tests. Apart from clonal changes, the polyclonal background of pathogenic effects responsible for the disease should be considered. The techniques for the demonstration mostly include all antibodies with an identical specificity rendering only one result. When the test system contains different antigenic structures to antibodies produced by different clones, each system separately yields an unknown diversity of affinity and concentrations, called avidity. Thus it is of substantial importance whether monoclonal or

1.3 Scope of tests

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3

polyclonal sera are used. Of course, this in turn depends on the analytical design as well as the diversity of antigens. These factors must be respected when autoimmune diagnostics for autoantibody detection are used routinely. The never-ending progress in technology causes changes that require amendments to provide standards optimally adapted to this development.

1.3 Scope of tests Diagnostic tools used to detect an autoimmune disease cannot be separated from clinical findings. There is no single laboratory test to diagnose an autoimmune disease in individuals without clinical signs and symptoms. Thus each laboratory test requires a specific antigen depending on symptoms or disorders pertinent to a disease or syndrome. In this context conformity and conformation as well as shape and appearance of the reacting agents are used to test for the presence of membrane-bound antibodies. Sometimes testing is extented by the search for free specific antibodies in the circulation. Pathogenic immune reactions against particular tissues often require a biopsy to produce a tissue sample to demonstrate at the side of since a specific antibody may be totally absorbed. This phenomenon explains that, depending on antigen availability and antibody production, specific autoreactive antibodies cannot be found in a small population of patients. In contrast, another small population may show antibodies without symptoms. A rare situation rises from the complete absence of the autoantigens. This is caused by its complete elimination following destructive inflammation or surgical removal as in autoimmune thyroiditis. In this case, autoantibodies may accumulate up to high titers. Besides techniques specific in terms of immunologic aspects, there are tests that render associated immunological data. For example, nonspecific elevation of immunoglobulin levels in the serum is found in many autoimmune diseases based on general immune activation, especially in rheumatic autoimmune inflammation and in autoimmune hepatitis. Finally, concomitant systems, such as the complement system, are involved in the pathogenesis of autoimmune disease. In hemolytic diseases, the measurement of the total complement activity – or of singular components – is useful because of the increased consumption of complement factors, whereas in SLE there is in addition a substantial deficiency of singular components of complement. Of note, similar alterations may be observed in liver diseases when the hepatic synthesis primarily is reduced. Immunogenetic markers including MHC or HLA are of highest importance in allogeneic histocompatibility. They may indicate a certain probability for the existence of an autoimmune disease. Genetic testing of these factors allows a clear discrimination of positive and negative results never changing during lifetime. In conclusion, laboratory tests have to be conducted under strictly observation of all technical and biological requests. The interpretation of the test results, however, considers the current clinical situation of the patient.

1.3.1 Procedure The diagnostic approach in the diagnostic process should follow general rules. In the beginning, screening tests such as the detection of all antinuclear antibodies (ANA)

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1 General aspects of autoimmune diagnostics

whereas tests for singular specifities, including dsDNA, antiScl70 antibodies and others, are used later in the diagnostic process. Depending on the disease and its typical major antigen this procedure may be modified. Factors influencing the decision how to proceed in the diagnostic approach are efforts, required time, urgency, availability of probes, condition of the patient and costs.

1.3.2 Factors influencing the results Several factors are able to change the results. They may arise from variabilities in either the immune system of the individual patient or the technique to detect the immune reaction. When the influence is restricted to a quantitative change, it may become important only as far as results are within a range, where a cutoff discriminates between positive and negative. When the influence is qualitative the results may differ in the whole range of the readings.

1.3.2.1 Age and sex Levels of total immunoglobulins change during lifetime. The normal serum level is reached in youth between the 8th and 15th year and usually remains stable during the rest of the life. In older individuals there is a trend to a slight increase. Due to a decrease of albumin the fraction of immunoglobulins is increasing. Only IgE levels continuously decrease in adults. All of these changes are within a range without relevance for diagnosis. Interestingly autoantibody can be found in the serum of healthy elder individuals. Generally speaking, in healthy individuals IgM-rheumatoid factor is never found in the age below 20 years whereas it can be detected in >20% in the age of 80 years. This is the same regarding cold-agglutinins and antinuclear factors at a lower level. Between male and female patients there is no significant difference.

1.3.2.2 Other diseases The distribution of body-fluids, regarding the proportion within the circulation and the residual body, can also influence the concentration of serum proteins. This is reflected by a change of total protein or of albumin. Loss of proteins via the intestine or the kidneys may cause a decrease of immunoglobulins in the serum.

1.3.2.3 Exhaustive exercise Immunoglobulins decrease during exhaustive exercise. This may be caused by an altered distribution of liquids. By contrast, this effect may be reduced by exsiccation. Recovery begins several hours after the end of the exercise and is accelerated by the uptake of liquids.

1.4 Therapy Medical therapies, especially immunosuppressive drugs, may also affect diagnostics in autoimmune diseases. Antimetabolites and alkylants inhibit the cellular metabolism. This effect corresponds with the metabolic activity influencing the cellular reproduction

1.5 Timing of investigations

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more than the synthesis of proteins. Since plasma-cells do not proliferate, there is only a marginal reduction in antibody production regarding the IgG- or IgA-type. Immunophyllins like cyclosporine inhibit the first step of the immune response; therefore these drugs may also reduce IgM-levels. Monoclonal Anti-CD20 is directed against B-cells causing a slow and retarded decrease of all immunoglobulins. Generally speaking, the decrease depends from type, dose and time of any application. Changes of immunoglobulin-levels also are found in malignancy. However, because of malnutrition on the one hand and of displacement of healthy cells by the malignant population on the other hand low levels of antibody are often found. Transfusion of whole blood and immunoglobulins causes an increase of immunoglobulin levels in the serum. Pathogenic factors, however, may even fall due to dilution. This effect becomes significant upon repeated transfusions within a short period of time. Radiation therapy may also reduce the activity of antibody-producing cells. This depends on the type and number of applications in accordance as well as with the irradiated area containing lymphatic tissue. Splenectomy is of minor importance. There are no significant changes in the long run. Summing up there are several situations which change the level of the immunoglobulins in the whole body and in the circulation. These changes however, are seldom of relevance for the interpretation of autoantibody levels.

1.5 Timing of investigations Since autoimmune diseases are self-perpetuating the repetition of investigations is not necessary. There may be no need to assure when the diagnosis is confirmed. Controls, however, may help to follow the efficiency of immunosuppressive therapy and the course of the disease. Sinking titers of autoantibody may indicate a reduced activity of the pathogenic process. But even in complete remission normally autoantibody can be detected.

1.6 Storage and dispatch In the majority of autoimmune diagnostic procedures, blood is analyzed. Transportation by postal service requires the separation of serum for the detection of antibodies. For antibody diagnostics, serum can be stored at −20˚C for months. Special treatment is necessary in particular for the detection of cold agglutinins and tests using erythrocytes or platelets. Testing of leukocytes and thrombocytes requires special solutions and temperatures. This should be discussed with the analyzing laboratory.

References 1. Delves PJ, Martin SJ, Burton DR, Roitt IM. Essential immunology, 11th ed. Blackwell Publishing. London. 2006;111–154. 2. Murphy K, Travers P, Walport M. Janeways immunobiology, 7th ed. Garland Science. Taylor & Francis Group. London. 2008;930–950.

2 Rheumatoid arthritis Rudolf Mierau and Ekkehard Genth

2.1 General aspects and clinical features Rheumatoid arthritis (RA) is the most common inflammatory musculoskeletal disease. It affects about 0.5% to 1% of the population and frequently leads to structural and functional damage, impairment of daily life activities, and loss of quality of life (1). Polysynovitis of joints, tendon sheaths, or bursae is the most common musculoskeletal manifestation, causing pain, stiffness, swelling, and loss of strength and motility. Synovitis in RA mostly has a symmetrical distribution involving the wrist, the metacarpophalangeal and proximal interphalangeal joints of the hands (uFig. 2.1), and the ankle and forefoot. Generally any peripheral joint may be affected as well as the joints of the upper cervical spine. Inflammation frequently leads to cartilage and bone destruction of affected joints

Fig. 2.1: Symmetrical swelling of the wrist, metacarpophalangeal joints, and proximal interphalangeal joints in RA.

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(uFig. 2.2) and to local and systemic bone loss. In principle RA is a systemic autoimmune disease. Extra-articular manifestations like anemia, rheumatoid nodules, serositis, vasculitis or accelerated atherosclerosis with cardiovascular manifestations may occur and are associated with increased mortality. In the early phases of the disease, diagnosis often is hampered by the fact that initial symptoms are equivocal and of low specificity. Early and correct diagnosis of RA is of great importance, however, in order to initiate early and effective treatment, which may not only suppress inflammatory activity but also prevent damage and improve outcome (2). New criteria for the classification of RA were published by the American College of Rheumatology (ACR) and the European League Against Rheumatism (EULAR) in 2010 (3). Although primarily developed as a basis for the recruitment of patients into clinical trials, these criteria are often used as a diagnostic tool for individual patients. The new ACR/EULAR criteria for RA redefine the current paradigm of RA by focusing on features at earlier stages of disease, which are associated with persistent and/or erosive disease, rather than defining the disease by its late-stage features (e.g. joint erosions, rheumatoid nodules) (4,5). Important early features are arthritis, especially of the small joints, disease duration of more than 6 weeks, presence of rheumatoid factor (RF) and/or anticitrulline antibodies and elevated erythrocyte sedimentation rate (ESR) or C-reactive protein (uTab. 2.1). The disease often does not start with the polyarticular pattern of joint involvement typical of full-blown RA but with oligoarticular or, in rare cases, even monoarticular patterns (i.e., with involvement of only a few or in extreme cases only one joint) instead.

Fig. 2.2: X-ray of the hands, showing destructive changes in the wrists and metacarpophalangeal joints as well as arterial calcifications in a patient with RA.

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2.2 Diagnostic workflow

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Tab. 2.1: Classification criteria for RA of the American College of Rheumatology and the European League against Rheumatism (3); target population: patients who have at least one joint with definite clinical synovitis (swelling) and whose synovitis is not better explained by another disease Classification criteria

Score1

A. Joint involvement 1 medium/large joint

0

2–10 medium/large joints

1

1–3 small joints

2

4–10 small joints

3

> 10 joints (at least one small joint)

5

B. Serology Neither RF- nor ACPA-positive

0

Low positive RF or low positive ACPA

2

High positive RF or high positive ACPA

3

C. Duration of synovitis < 6 weeks

0

≥ 6 weeks

1

D. Acute-phase reactants C-reactive protein and erythrocyte sedimentation rate normal

0

C-reactive protein or erythrocyte sedimentation rate abnormal

1

1. Scores of categories A to D are added; the highest score of each category is counted; up to 10 score points are possible; a score of ≥ 6 points is needed for classification as definite RA.

The following features are regarded as having high prognostic value with regard to persistence of arthritis (1,2,6,7,8,9,10): • • • • • •

Longer disease duration (several months) Many actively inflamed joints Female sex Rheumatoid factor (RF) Anticitrullin-peptide antibodies (ACPA) High level of inflammatory activity

2.2 Diagnostic workflow As shown in uFig. 2.3, arthritis of two or more joints is the clinical starting point, potentially leading to a diagnosis of RA. Arthritis in this case means a “soft“ joint swelling, caused by effusion and/or synovial thickening, observed by the physician during physical examination of the patient. The swelling has a soft, pasty consistency and does not resist compression of the palpating finger; its content can be pressed into the contralateral part of the synovial cavity (11,12). This finding must be distinguished from

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Fig. 2.3: Flow scheme for diagnosis of RA.

a “hard” swelling because of bony enlargement in osteoarthritis and some other arthropathies, from extra-articular fat pads, and from periarticular swelling or edema. For joints that cannot be appropriately palpated and inspected because of their anatomical position (e.g., hip or sacroiliac joints), a suitable imaging technique like ultrasound or magnetic resonance imaging should be used. Starting from this clinical finding, further diagnostic steps are initiated that are depicted in uFig. 2.3. On the clinical side, findings typical for early RA are morning stiffness in and around the joints lasting for at least 1 hour before maximal improvement as well as the number and distribution pattern of affected joints: for the majority of the patients the onset is polyarticular (five or more joints involved). Oligoarticular onset (two to four joints) is not uncommon; a monoarticular one, however, is rare. Affection of the proximal interphalangeal (PIP), metacarpophalangeal (MCP), and metatarsophalangeal (MTP) joints (often symmetrical) is a further typical RA feature, especially as the first manifestation. Other joints commonly involved (also often symmetrically) are the wrist, knee, ankle, elbow, and shoulder joints. A distinctive affection of the distal finger or toe joints is unusual. By physical examination, other inflammatory musculoskeletal (e.g., tenosynovitis, bursitis) or extramusculoskeletal features like subcutaneous rheumatoid nodules or carpal tunnel syndrome (12,13) may also frequently be detected. The systemic character of RA is expressed by anemia, Raynaud’s phenomenon, vasculitis, Sjo¨gren’s syndrome, Felty’s syndrome, or cardiovascular, pulmonary, neurological, or ocular manifestations in some patients. Laboratory findings contribute to the diagnosis of early RA mainly in two areas: by measuring systemic inflammation and detecting autoantibodies typical for RA.

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To assess inflammatory activity, the erythrocyte sedimentation rate (ESR) and/or C-reactive protein (CRP) is measured. Normal values for these analytes lower the probability of RA but do not exclude it, since a considerable proportion of RA patients have ESR and/or CRP values in the normal range at their first presentation (14,15). During the course of the disease, with continuing inflammation, hypochromic anemia is often observed. In this case drug-induced gastrointestinal blood loss should be excluded. To use autoantibodies as diagnostic aids for RA, measurement of RF and antibodies against citrullinated peptides or proteins (ACPA) is currently recommended. Each test should be performed once. A negative result in both assays makes RA unlikely, although it does not exclude the disease completely. A positive result in both tests raises the probability of RA to about 90% to 100%. Discordant results for RF and ACPA are ambiguous but also raise the a priori probability for RA. Autoantibodies are determined in serum or plasma. The examination of synovial fluid for autoantibodies does not result in increased sensitivity. Analysis in synovial fluid can be impaired because of high sample viscosity. Since none of the findings from clinical and laboratory investigations described above are completely specific for RA, differential diagnosis is very important, particularly in early disease phases. The list below shows arthritic diseases relevant in this context that should be excluded if necessary (11,12,13). Disorders relevant for differential diagnosis of RA (for adults): • • • • • • • • • • • • • • •

Arthritis in connective tissue diseases* Arthritis in systemic vasculitides* Arthritis in polymyalgia rheumatica R3SPE syndrome (remitting seronegative symmetric synovitis with pitting edema) Undifferentiated spondyloarthropathies Psoriatic arthritis Enteropathic arthritides (in Crohn’s disease, ulcerative colitis) Post-/parainfectious (postenteritic or posturethritic) reactive arthritis, rheumatic fever; borreliosis, hepatitis B or C, parvovirus B19, among others) Polyarticular gout Pseudorheumatoid form of chondrocalcinosis Activated osteoarthritis Sarcoidosis* Infectious endocarditis* Hemochromatosis Diabetic arthropathy

* Rheumatoid factors are also common in these disorders.

2.2.1 Properties and diagnostic roles of RA-typical autoantibodies Rheumatoid factors (RFs) are the classic serological hallmark of RA. They are by definition antibodies directed against the Fc portion of immunoglobulin G (of human or other mammalian origin). RFs can belong to any immunoglobulin class (isotype), but the routinely determined RFs are mainly IgM and IgA. They can be found in 70% to 90% of the RA patients but are of low specificity for this disease in differential diagnosis

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Tab. 2.2: Prevalence of rheumatoid factors Disease

Frequency (%)

Rheumatoid arthritis

70–90

Sjo¨gren’s syndrome

75–95

Mixed connective tissue disease

50–60

Systemic lupus erythematosus

15–35

Systemic sclerosis

20–30

Mixed cryoglobulinemia type II

100*

Systemic vasculitides (Panarteriitis nodosa, Wegener’s granulomatosis, and others)

5–20

Chronic sarcoidosis

5–30

Healthy persons < 50 years old

70 years old

10–25

*Monoclonal IgM rheumatoid factors. In addition RFs are found – in various frequencies and mostly in low concentrations – in diverse nonrheumatic inflammatory, infectious, and neoplastic disorders.

because they may also occur in several other rheumatic (uTab. 2.2) and nonrheumatic (inflammatory, infectious, and neoplastic) disorders (16,17). In many rheumatic diseases relevant for the differential diagnosis of RA (psoriatic arthritis, other spondyloarthropathies, reactive arthritis, gout, osteoarthritis), however, RFs are not more frequent than in healthy persons. Several assay systems for the detection of RFs are available and well established, from simple qualitative agglutination assays to quantitative measurements by, for example, enzyme immunoassay or nephelometry. On the contrary, assay systems for ACPA assays (usually as enzyme immunoassays or other binding test systems) have become commercially available during the past decade. These binding assays with synthetic antigens had been preceded by several earlier systems, namely the measurement of antiperinuclear factor (18), antikeratin (19), anti-Sa (20), and antifilaggrin (21,22). All these assay systems detect, as was realized from about 1998 on (23,24,25), antibodies against proteins or peptides in which arginine residues are converted to citrullin by the enzyme peptidylarginine deiminase (PAD) (uFig. 2.4). Schellekens et al. clarified that RA patients’ sera react with several epitopes containing citrulline residues (with fine specificity patterns differing from patient to patient) (23) and used a first-generation assay (CCP1) with a single filaggrinderived citrullinated peptide (cfc1), which was converted to a cyclic molecule by two serine-to-cystein-substitutions (cfc1-cyc) (26). By selecting better epitopes from peptide libraries, the second-generation CCP assay (CCP2) was developed, made commercially available, and extensively used for diagnostic studies (27,28,29,30). Several licensed assay kits for this analyte are on the market (17,31). Recently a third-generation CCP assay (CCP3) has been marketed (32,33,34,35,36,37,38,39,40,41,42,43,44). In addition, many assays (ELISA, line-assay, and other binding assays) using alternative citrullinated peptides or proteins have been developed and increasingly are being put to market; therefore, the term anti-CCP has been replaced by ACPA. Among the alternative

2.2 Diagnostic workflow

冷

13

Fig. 2.4: Conversion of arginine into citrulline residues in polypeptide chains by the enzyme peptidylarginine deiminase.

citrullinated polypeptides are filaggrin (32,45,46,47,48,49,50,51,52), fibrinogen (38,53,54,55,56,57,58,59,60,61), collagen (62), mutated vimentin (32,39,41,43,51, 52,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81), as well as several alternative peptides (51,56,61,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98). Up to now, for many of these assays, sensitivity and specificity for RA are in comparable ranges; their results correlate well but not perfectly with each other (31). Usually, greater sensitivity comes with lower specificity, and vice versa. Efforts to improve the ACPA assays in terms of diagnostic accuracy, prognostic value, or correlation with disease activity seem justified since CCP is a purely synthetic construct and not a “real” autoantigen (99), although, strictly speaking, this is likewise true for many xeno- and/or recombinant antigens succesfully used in the diagnosis of human autoimmune diseases. ACPA are similar to RF in their diagnostic sensitivity for RA but are superior by far in terms of specificity. This is true also for elderly onset (100) polyarthritis. ACPA-positive patients with early undifferentiated arthritis have a markedly elevated risk for progressing to RA (8,9,10, ,55,101,102,103,104,105). uTab. 2.3 lists the published prevalence data for anti-CCP in different diseases. Diagnostic specificity is highest (>99%) when compared to healthy controls (some of which even might be pre-RA patients since ACPA [and RF] have been shown to precede onset of disease symptoms in most cases, by up to several years [106,107,108,109, ]). In certain inflammatory rheumatoid diseases besides RA, however, ACPA prevalences of about 5–10 % are found. When patients with these diseases are stratified according to their ACPA status, those positive for ACPA more often have features related to RA: synovitis in systemic sclerosis (36,110,111,112,113,114), Sjo¨gren’s syndrome (114,115,116,117,118,119), or MCTD (114,120); erosive arthritis in systemic lupus erythematodes (121,122,123,124,125, 126,127); erosive, often symmetric, polyarthritis and rheumatoid factors in psoriatic arthritis (128,129,130,131,132,133,134); and polyarticular disease, often erosive, with relatively high age at onset and rheumatoid factors in juvenile chronic arthritis (37,135,136,137,138,139,140). In addition, also for non-RA patients, ACPA were reported to be associated with the RA typical genetic factor “shared epitope“

8,207

1765

2127

1008

211

429

267

171

1807

Early rheumatoid arthritis (duration less than 1 year)

Systemic lupus erythematosus

Sjo¨gren’s syndrome

Systemic sclerosis

Mixed connective tissue disease

Poly-/ Dermatomyositis

Systemic vasculitides

Cryoglobulinemia

Psoriatic arthritis

521

23,350

Rheumatoid arthritis

4.4

6.1

2.3

4.1

5.8

9.0

6.9

6.4

6.6

60.2

71.3

Frequency (%)

(10,28,29,30,49,50,57,74,138,141,194,202,203,206,207,213,226,229,246,248,253,264,276, 287,288)

(26,28,29,30,32,49,51,57,128,129,130,131,132,133,134,138,192,199,202,206,213,229,231, 253,264,266,287,288,296,297)

(200,210,212,295)

(26,28,49,50,57,111,138,194,202,208,213,217,229,237,253,276,290)

(23,26,28,32,65,120,138,163,192,194,201,205,207,213,217,226,229,253,286,294)

(30,32,49,50,111,120,138,163,192,201,202,205,213,217,226,229,248,264)

(23,26,28,32,36,50,57,74,110,111,113,120,138,163,192,194,201,202,205,206,207,213,217, 226,229,246,253,266,287,288)

(23,26,28,32,41,49,65,74,82,111,113,115,117,118,120,138,192,194,199,201,203,204,205, 206,207,208, ,211,213,217,226,229,248,253,287,288,289,290,291,292,293)

(10,23,26,28,29,30,32,41,49,50,57,74,82,111,120,122,123,136,138,163,192,193,194,201, 202,203, ,205,206,207,208, ,211,213,214,217,226,229,248,253,264,266,272,284,285, 286,287,288)

(9,14,26,28,30,32,49,74,106,130,153,161,170,194,196,199,201,202,203,217,229,230,260, 261,262,263,264,265,266,267,268,269,270,271,272,273,274,275,276,277,278,279,280, 281,282,283)

(14,26,28,29,32,36,41,50,51,57,64,65,82,86,100,111,113,120,123,136,138,139,141,144, 163,181,182,191,192,193,194,195,196,197,198,199,200,201,202,203,204,205,206,207, 208,209,210,211,212,213,214,215,216,217,218,219,220,221,222,223,224,225,226,227, 228,229,230,231,232,233,234,235,236,237,238,239,240,241,242,243,244,245,246,247, 248,249,250,251,252,253,254,255,256,257,258,259)

References

冷

Other spondyloarthropathies

Number of cases

Disease

Tab. 2.3: Prevalence of anti-CCP (the ACPA predominantly measured in studies up to now)

14 2 Rheumatoid arthritis

720

355

370

107

1442

138

7150

Osteoarthritis and other noninflammatory rheumatic diseases

Inflammatory bowel diseases

Autoimmune hepatitis

Autoimmune thyroid diseases

Infectious disease (without arthritis and without tuberculosis)

Tuberculosis

Healthy persons (elderly people included)

136

Palindromic rheumatism

72

281

Polymyalgia rheumatica

Crystal arthropathies

158

98

1023

Reactive arthritis

Borreliosis

Juvenile chronic arthritis

0.9

26.1

1.7

0.9

8.9

2.0

4.7

2.8

52.9

2.5

3.8

5.1

8.3

(14,26,28,41,50,51,65,74,100,107,110,122,130,131,136,137,138,144,153,154,192,194,198, 201,203,205,214,215,219,223,226,228,229,236,237,241,246,248,259,266,272,275,286,290, 292,295,300,301,305,306,307)

(26,74,144,194,305)

(26,28,51,57,74,138,154,192,194,200,205,208,210,212,229,236,252,256,286,295,303,304)

(28,32,51,192,205)

(194,205,210,236,302)

(10,28,111,194,228,286)

(10,26,28,29,30, ,41,49,50,57,65,194,201,208,226,229,231,247,287,288,300,301)

(30,49,50,57,138,207,229,287)

(141,142,213,235)

(51,57,100,111,138,192,199,213,218,226,264,287,299)

(26,28,30,50,57,138,194,201,213,226,253,264,286)

(26,28,30,51,192,207,264)

(29,30,37,50,135,136,137,138,139,140, ,192,193,202,213,226,229,253,298)

2.2 Diagnostic workflow

冷 15

16

冷

2 Rheumatoid arthritis

(116,119,122,129,137). ACPA might be a marker not for what we call RA but for arthritis in patients carrying the RA-predisposing genetic factors (116). Most patients with palindromic rheumatism are thought to have a variant in disease course of RA in which ACPA have a prognostic value for progression to full-blown RA (141,142). Anti-CCP in autoimmune hepatitis, in tuberculosis and in SLE without deforming/erosive arthritis seem to be “false positives” since they mostly are not citrulline dependent (125,143,144,145). Determination of both RF and ACPA for diagnosis of early RA, as outlined above in the diagnostic workflow, is justified because in spite of some correlation between the two autoantibody systems, there is a certain percentage of RA patients scoring positive only for one of them (uTab. 2.4a and 2.4b). Considering RA when at least one of the two autoantibodies (rather than both) is detected therefore improves sensitivity, although at the expense of diagnostic specificity. Even in very sensitive ACPA assays some RA patients remain negative. Grouping RA patients into ACPA positives und ACPA negatives uncovers differences in addition to ACPA (146,147,148) (uTab. 2.5). Although both subsets have considerable hereditary components, their genetic risk factors differ. Environmental contributions and histopathological features might also differ between the two groups. Clinically they are indistinguishable in the beginning, however the ACPA positives on average suffer from a more severe, more rapidly progressing disease. Tab. 2.4a: Combinations of autoantibody results in patients with rheumatoid arthritis, Patients with established RA. Publication

RF + ACPA + [%]

RF + ACPA – [%]

RF – ACPA + [%]

RF – ACPA – [%]

Schellekens et al. 1998 (23)

65

18

11

6

Bizarro et al. 2001 (192)

36

26

5

33

Zeng et al. 2003 (194)

37

21

10

32

Lee & Schur 2003 (29)

57

15

10

18

Bas et al. 2003 (306)

48

25

8

19

Dubucquoi et al. 2004 (82)

68

5

9

17

Lopez-Hoyos et al. 2004 (100) 54

12

11

23

Vallbracht et al. 2004 (198)

51

15

13

21

Irigoyen et al. 2005 (308)

63

10

6

21

Kwok et al. 2005 (138)

46

13

9

32

Fusconi et al. 2005 (210)

57

26

2

15

Sauerland et al. 2005 (208)

65

4

9

22

Choi et al. 2005 (50)

68

12

5

15

Mewar et al. 2006 (309)

63

5

18

14

Hill et al. 2006 (57)

72

8

9

11

Korkmaz et al. 2006 (219)

70

12

5

13

Agrawal et al. 2007 (221)

74

8,5

8,5

9

2.2 Diagnostic workflow

冷

17

Tab. 2.4b: Combinations of autoantibody results in patients with rheumatoid arthritis; Patients with early RA (disease duration < 1 year in most publications). Publication

RF + ACPA + [%]

RF + ACPA – [%]

RF – ACPA + [%]

RF – ACPA – [%]

Goldbach-Mansky et al. 2000 (191)

36

30

5

29

Schellekens et al. 2000 (26)

39

15

9

37

Kroot et al. 2000 (260)

58

22

8

12

Jansen et al. 2002 (261)

33

13

9

45

Rantapa¨a¨-Dahlquist et al. 2003 (106)

58

15

12

15

Saraux et al. 2003 (263)

49

24

21

6

Dubucquoi et al. 2004 (3

−2

2

+3

(x-fold above normal)

1.5–2

+2

1–1.5

+1

1:80

+3

(titers)

1:80

+2

1:40

+1

15 10–15 >17 12–17

9.1 General aspects and clinical features

冷

105

International Autoimmune Hepatitis Group, (IAIHG) in 2008 (uTab. 9.2) in order to facilitate the diagnosis (14). The score calculated by these criteria displays a sensitivity of 88% and a specificity of 97% for probable AIH and a sensitivity of 81% with a specifity of 98% in diagnosing definite AIH in a collective of adult AIH patients (mean age: 49 years, range: 34–59 years). Viral hepatitis B and C should be ruled out by testing for HBsAg (+ anti-HBc) and anti-HCV. Alcohol consumption, intake of hepatotoxic medication, and hereditary liver diseases (Wilson’s disease, hemochromatosis, alpha-1 antitrypsin deficiency) should be excluded. The first hint of the presence of AIH is the demonstration of elevated gamma globulins on serum electrophoresis or, alternatively, quantitative IgG serum level, which is probably the cheapest screening test for AIH. However, around 5% to 10% of AIH patients do not display elevated gamma globulins at initial diagnosis. Some have a relative elevation of their gamma globulin levels within the wide physiologic range, which becomes apparent only when, upon immunosuppression, the gamma globulin levels fall from the upper normal range to the lower normal range. Serum lipids should be tested and ultrasound should be performed, since fatty liver disease is an important consideration in the differential diagnosis. Quantitative IgA, IgG, and IgM levels are very helpful diagnostic markers. In AIH there is typically a selective elevation of IgG, with normal levels for IgA and IgM. Elevated IgA hints at a toxic cause (e.g., alcohol) of the liver disease, whereas elevated IgM is a feature of biliary disease such as primary biliary cirrhosis or primary sclerosing cholangitis. Both of these conditions can be associated with AIH (so-called overlap syndromes, as mentioned below).

Tab. 9.2: Simplified diagnostic criteria for autoimmune hepatitis Parameters

Points

1. Autoantibodies

ANA or SMA or LKM >1:40 ANA or SMA or LKM >1:80 SLA/LP positive (>20 units)

1 2 2

2. IgG (or gamma-globulins)

Upper normal limit >1.10 times normal limit

1 2

3. Liver histology*

Compatible with AIH Typical for AIH

1 2

4. Absence of viral hepatitis

Yes No

2 0

ANA, antinuclear antibody; SLA, soluble liver antigen; IgG, immunoglobulin G; AIH, autoimmune hepatitis. Adapted from reference 14. *Typical: (a) Interface hepatitis, lymphocytic/lymphoplasmacytic infiltrates in portal tracts and extending in the lobule; (b) emperipolesis (active penetration by one cell into and through larger cell); (c) hepatic rosette formation. Compatible: Chronic hepatitis with lymphoctic infiltration without features considered typical. Atypical: Showing signs of another diagnosis, like non-alcoholic fatty liver disease (NAFLD). Definite AIH: ≥7; probable AIH: ≥6.

106

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9 Autoimmune liver disease

About 80% of all AIH patients have pathologic titers of at least one autoantibody − in North American adults, up to 96% (15). Antinuclear antibodies (ANAs) and antibodies to smooth muscle antigen (SMA), typical for type 1 AIH, occur in 40% to 50% of the patients each, sometimes in combination, and antibodies to soluble liver antigen (SLA/LP), typical for type 3 AIH, present in about 20% of patients. These antibodies constitute the conventional serologicl repertoire for the diagnosis of AIH. Antibodies to liver-kidney microsomal antigen (LKM) are uncommon (1%–3%) but delineate a separate disease subgroup, often called type 2 AIH, which is probably more prevalent in childhood, but often indicates severe disease. ANA, LKM-1, and SMA autoantibodies can be detected on indirect immunfluorescence of tissue sections (mostly composite sections of stomach, kidney, and liver from rodents) or on Hep-2 cells (16), available from numerous suppliers; this will, at the same time, detect antimitochondrial antibodies (AMAs) as the characteristic test for primary biliary cirrhosis. A serum dilution of 1:80 is used in screening for these antibodies. In case of a positive result, the test is repeated using a geometric series of dilutions according to the strength of the primary fluorescence (1:160−1:5,120). Composite sections of different tissues and Hep-2 cells contain a variety of detectable autoantigens in the nucleus and cytoplasm. Autoantibody fluorescence patterns are subdivided into basic patterns with either nuclear, cytoplasmic, or mixed fluorescence. uTab. 9.3 shows typical nuclear and cytoplasmic fluorescence patterns on Hep-2 cells of autoantibodies, possible antigens, and associated diseases. SLA/LP antibodies do not show up on immunofluorescence and are therefore missed by routine testing. These antibodies should be looked for by specific immunoassays such as enzyme-linked immunosorbent assay (ELISA) and/or immunoblot. Specific immunoassays are also used for F-actin SMA-autoantibodies, LC-1, LKM-3, pANCAs, or AMA-M2 (the latter for the diagnosis of PBC) in case of negative fluorescence testing or fluorescence patterns that cannot be characterized more closely. Tab. 9.3: Allocation of Hep-2 cell immunofluorescence patterns, underlying autoantigens, and possible diseases Pattern

Autoantigens

Possible disease

dsDNA, nucleosomes, histones

SLE, AIH

Nuclear patterns Homogenous Fine granular

Ro/SS, La/SS-B, Ku

SS, AIH

Coarse granular

U1-RNP, Sm

MCTD

Nucleolar

Scl-70, Fibrillarin, Th/To, PM-Scl

SS, PM, DM

Centromere

CENP-A, B, C

SS, PBC

Nuclear dots

Sp 100

PBC

Cytoplasmic patterns Homogenous

Rib-P

Granular

Jo-1

ASS

Mitochondrial

AMA-M2

PBC

9.2 Primary sclerosing cholangitis

冷

107

Whereas ANA, SMA, and LKM can also be found in some patients with other liver diseases and nonhepatic autoimmune diseases, SLA/LP antibodies are highly specific for AIH and thus diagnostic in the patients in whom they can be demonstrated (17). This seems also to be true for antibodies to double-stranded DNA, which are detectable in 15% of patients and for which the only differential diagnosis is systemic lupus erythematosus (SLE). Antibodies to cyclic citrullinated peptides (anti-CCPs) are found in 9% of patients with AIH (18). No or low autoantibody titers do not exclude the diagnosis of AIH, nor do high titers in the absence of other criteria prove the diagnosis (15). Liver biopsy at presentation is recommended to establish the diagnosis and for grading of histologic activity, fibrosis staging, and to aid treatment decision and initiation. Interface hepatitis, bridging or multiacinar necrosis, and the presence of a lympho-/ plasmacellular infiltrate are typical for AIH (uFig. 9.1). Liver biopsy is also performed in order to exclude important differential diagnoses such as drug-induced liver disease or nonalcoholic steatohepatitis (NASH). As mentioned above, about one quarter of all patients with AIH already have cirrhosis at the time of diagnosis (16). Cirrhosis is often macronodular, which explains why in many of these patients cirrhosis may be missed because of the sampling error (biopsy of a large regenerative nodule without fibrous septa). Some authors therefore favor laparoscopy at initial diagnosis, which can now be performed with minimal invasiveness (19,20).

9.1.2.1 Overlap syndromes with PBC or PSC About 10% to 20% of AIH patients at some stage of their disease have overlapping features, with either primary sclerosing cholangitis (PSC) (21) or more commonly primary biliary cirrhosis (PBC) (22). In these patients management should be primarily directed toward the AIH component of their disease, as this is the condition leading most rapidly to liver destruction and cirrhosis; it can also be treated effectively. In overlap patients it appears sensible to add ursodeoxycholic acid (UDCA) to the treatment schedule, even though the evidence for this approach is weak. Patients with AIH/PSC overlap seem to run a less favorable course than patients with AIH only, whereas AIH/PBC overlap patients seem to have a similarly good prognosis.

9.2 Primary sclerosing cholangitis PSC is a chronic inflammatory cholestatic liver disease of unknown cause with a genetic predisposition and environmental factors also possibly playing a role. An increased prevalence of HLA class I A1 as well as HLA class II B8, DR3, and DR4 is observed in PSC patients (23). PSC mostly progresses to cirrhosis via fibrotic strictures and dilations of extra- and/or intrahepatic bile ducts, but the disease course is highly variable (24). Half of the patients develop dominant strictures of the large bile ducts with recurrent episodes of bacterial cholangitis (25). Most patients develop liver cirrhosis within 12 to 17 years after their first diagnosis irrespective of any medical treatment (26). The pathogenesis is not understood, autoimmune processes, toxic bile acids, or bacterial translocation of a “leaky” gut are the three leading hypotheses on the pathogenesis of PSC (27). Approximately 80% of patients have concurrent inflammatory

108

冷

9 Autoimmune liver disease

Fig. 9.1: a–b Autoimmune hepatitis (AIH) consistent with interface hepatitis, (a) plasma cell infiltration and (b) hepatic rosetting (cluster of hepatocytes surrounded by inflammatory cells, marked by an arrow.

bowel disease (IBD), specifically ulcerative colitis in most. PSC predisposes to hepatobiliary malignancies such as cholangiocarcinoma (3.3%–36.4% of the PSC patients), gallbladder carcinoma, and hepatocellular carcinoma as well as colorectal carcinoma in patients with concurrent IBD. Therefore an early diagnosis is important to allow for regular screening procedures. UDCA and endoscopic bile duct dilation can relieve symptoms and improve the liver enzyme profile. Orthotopic liver transplantation is the only potentially curative therapy.

9.2 Primary sclerosing cholangitis

冷

109

9.2.1 Epidemiology The prevalence of PSC in northern Europe is approximately 1 in 10,000; it decreases to 1 in 100,000 and 1 in 1 million in southern Europe and Asia. Median age at onset is 30 to 40 years, with a wide range from childhood to senescence. Some 70% of the patients are male.

9.2.2 Diagnosis The diagnosis of PSC is made on the basis of a predominant cholestatic liver enzyme profile (mostly alkaline phosphatase, gGT, and, in later stages, bilirubin,) indicating cholestasis, characteristic bile duct abnormalities in cholangiography (uFig. 9.2a), or liver biopsy (uFig. 9.2b) and by exclusion of secondary etiologies. A variant of PSC, ”small duct PSC,” starts with isolated changes of the small intrahepatic bile ducts. Then liver biopsy is essential to make an early diagnosis, since involvement of the larger bile ducts often occurs at later stages after disease progression. No specific clinical, biochemical, or histologic finding enable the diagnosis of PSC. The European Association for the study of the liver (EASL) and the American Association for the Study of Liver Diseases (AASLD) have published guidelines for the diagnosis of PSC (uTab. 9.4) (28,29). PSC is associated with multiple autoantibodies; most common are atypical perinuclear antineutrophil cytoplasmic antibodies (pANCA: 33%–87%), ANAs: 7%–77% and SMA autoantibodies (13%–20%). A pathogenic role for these autoantibodies is uncertain (30). Imaging hallmarks in PSC are multiple segmental intra- and extrahepatic strictures, diverticular pouchings, beaded ducts, and a pruned appearance of the biliary tree in ERC (uFig. 9.2). MRC also seems to be sensitive in the evaluation only up to secondor third-order intrahepatic branches and therefore misses early changes, particularly in nondilated ducts. The combination of MRC and MRI also provides information about the abdominal status and potential complications, such as cholangiocarcinoma (CCA). In early detection of CCA, the role of tumor markers (CA19–9 and CEA) is limited. The diagnostic accuracy in the differentiation between malignant and benign strictures seems better in MRC than in ERC (31). Positron emission tomography (PET) and PET-CT can not distinguish malignant from benign stenoses but is comparable to CT, MRI/MRCP in staging of CCA patients preoperatively (PET-CT: sensitivity 84%, specificity 79%, PPV 92%, and NPV 60%). Only regional lymph node metastases (75.9% vs. 60.9%, P = 0.004) and distant metastases (88.3 vs. 78.7%, P = 0.004) can be assessed with a higher accuracy by PET-CT than CT alone (32). Cholangioscopic characterization of bile duct stenoses has been shown to provide the highest detection rate of malignant stenoses (sensitivity 92%, specificity 93%, positive predictive value (PPV) 79%, and negative predictive value (NPV) 97%) (33). Recently a new variant of autoimmune cholangitis has been reported. Immunoglobulin 4−associated cholangitis (IgG4-SC) is characterized by sclerosing inflammation and infiltration of IgG4-positive plasma cells around intrahepatic and/or extrahepatic bile ducts. It is not clear whether IgG4-SC represents a distinctive disease or another variant of PSC. The association with autoimmune pancreatitis and other extrahepatic immune conditions suggests a separate disease entity. The bile duct changes also

110

冷

9 Autoimmune liver disease

Fig. 9.2: a–b Primary sclerosing cholangitis (PSC) in ERC with bile duct strictures and stenoses (a). Liver histology in primary sclerosing cholangitis (PSC) with concentric periductal fibrosis of bile ducts and surrounding cuffs of mixed inflammatory cells (b).

tend to look different, with predominant hilar continuous stenoses. The Mayo Clinic subsequently proposed HISOR-t criteria for diagnosis of IgG4-SC (uFig. 9.3) (34). Although steroid therapy is ineffective in PSC, IgG4-SC responds dramatically to steroids or immunosuppression.

9.3 Primary biliary cirrhosis

冷

111

Tab. 9.4: Summary of the current EASL and AASLD practice guidelines on primary sclerosing cholangitis (PSC) (28,29)* Practice point

EASL

AASLD

MRC (initially)

+

+

ERC (initially)

0, if indicated

0, if indicated

Liver biopsy (adults)

0, if M/ERC regular

0, if M/ERC regular

Liver biopsy (children)

+

+

Antibiotic prophylaxis (ERC)

+

−

Long-term antibiotic therapy

−

0, if recurrent bacteremia

Endoscopic treatment balloon dilatation

+

+

Endoscopic treatment Stenting

+

+

UDCA treatment

−

−

UDCA chemoprevention

0, long-term IBD, CRRF

−

Treatment of AIH/PSC overlap immunosuppression

+

+

Treatment of AIH/PSC overlap UDCA

+

−

LTX

+

+

Surveillance colonoscopy

+, 1–2 years in adults

+, 1–2 years in adults

Surveillance ultrasound

+, annually

+, annually

CCA surveillance

−

−

*Key: +, recommended; 0, recommended with limitations; –, not recommended or no recommendation made. MRC = magnetic resonance cholangiography; ERC = endoscopic retrograde cholangiography.

9.3 Primary biliary cirrhosis Primary biliary cirrhosis (PBC) is a chronic cholestatic autoimmune disease affecting small interlobular bile ducts in the liver; it has a slowly progressive course. PBC has a clear female preponderance (being almost 10 times as common in women versus men); it affects women with a prevalence of 1 in 2,000. Half of the patients are asymptomatic at first diagnosis because PBC is now diagnosed earlier than in the past, but fatigue, pruritus, and jaundice may be reported. Multiple genetic factors and environmental triggers (e.g., infections, chemical xenobiotics) that may affect the immune system are involved in the pathogenesis of PBC. A high concordance in monozygotic twins and a relative risk of about 5% in first-degree relatives is reported. UDCA, when administered at doses of 13 to 15 mg/kg per day, may lead to a normal life expectancy for most of these patients. About one third may require additional medication or liver transplantation. Three major forms of PBC can be differentiated, the typical or classic form in about

112

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9 Autoimmune liver disease

Fig. 9.3: Diagnosis of IgG-4−associated cholangitits (IAC).

70% of the patients, with slow decline of small bile ducts and a parallel increase of fibrosis. A second form is characterized by features of AIH, so called “overlap syndrome” with an accelerated development of fibrosis. The third very rare form, a premature ductopenic variant, is the most severe form, with a rapid onset of ductopenia and icteric cholestasis, which progresses to cirrhosis within less than 5 years (35).

9.3.1 Diagnosis Most patients are asymptomatic at diagnosis. In those who are symptomatic, fatigue and/or pruritus occur in about 20% at initial presentation (36). Most asymptomatic patients will develop symptoms within 2 to 4 years, although one third will not develop any symptoms for many years (37). The vast majority (up to 80%) of PBC patients will complain of chronic fatigue during follow-up independent of the stage of disease. An association of fatigue with sleep disorders, orthostatic hypotension, and depression has been demonstrated (38). Pruritus occurs more often in PBC than in other cholestatic diseases and will develop in up to 70% of patients during the course of PBC. It can sometimes develop without an association with the severity of the disease. The major hallmark of PBC is a high titer of antimitochondrial antibodies (AMAs) in serum, especially AMA-M2 antibodies that react with the E2 component of pyruvate dehydrogenase (PDH-E2) of the mitochondria. AMA-M2 autoantibodies are pathognomonic for PBC, and AMA-positive individuals without cholestasis are expected soon to develop PBC (39). This high positive predictive value, resulting from a nearly 100% specificity, is associated with a 95% sensitivity of AMA-M2 autoantibodies. Around 5% of the patients with typical biochemical and histologic features of PBC are negative for AMA and its subtype AMA-M2, despite a characteristic T-cell response to PDH-E2 (40).

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Another typical serologic finding is the elevation of IgM, probably due to an abnormal chronic B-cell activation. Since high IgM levels are unspecific, this supports the diagnosis of PBC but is not used as a diagnostic criterion. In about one third of PBC patients, nonspecific antinuclear autoantibodies and/or SMA autoantibodies can be found, but they are of minor diagnostic relevance. Specific ANAs react with various antigens of the pore complex. gp210 and nucleoporin 62 present as perinuclear rims. Antigens of the nuclear body, such as sp100, present with multiple nuclear dots on indirect immunofluorescence and also display high specifities for PBC, up to 95%, but poor sensitivity. Specific ANAs can be useful markers in AMA-negative patients. Serum AP and gGT define a cholestatic enzyme pattern and are commonly elevated in PBC, whereas aminotransferases (ALT and AST, hepatitc enzyme pattern) are normal or only slightly elevated in early stages. An increase of bilirubin (mostly conjugated) can be observed in later stages of PBC, when fibrosis progresses to cirrhosis and platelets decrease. Liver dysfunction then appears, with reduced protein synthesis of, for example, cholinesterase, albumin, and coagulation factors as biochemical markers of liver cirrhosis. Serum cholesterol is often elevated, as in other cholestatic diseases, because of the presence of an abnormal lipoprotein LpX, consisting of phospholipids and free cholesterol. LpX, when regurgitated from the bile into the blood, bears apparently no atherogenic potential but is associated with the development of xanthomata and xanthelasma in PBC patients. A liver biopsy is mandatory only in AMA-negative PBC patients. However, liver histology is helpful for prognostic evaluation by assessing disease activity and fibrosis stage, the exclusion of other concomitant hepatic diseases, and for treatment strategy. Typical histologic features are: periportal inflammation with bile duct−infiltrating lymphocytes and plasma cells, focal destruction of the intralobular bile ducts within portal triads and compensatory bile duct proliferation, bile duct obliteration, and granuloma formation (uFig. 9.4). The staging systems used are Ludwig’s or Scheuer’s classification;

Fig. 9.4: Primary biliary cirrhosis (PBC), demonstrating nonsuppurative destructive cholangitis, lymphocyte infiltration in bile ducts, and ductal proliferation.

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in both systems stage 3 represents fibrous septa and scarring, whereas stage 4 represents cirrhosis (41). Few differences are noted between stage 1 and stage 2, where Ludwig’s classification focuses more on histologic activity and Scheuer‘s describes more morphologic changes of the bile ducts. However, because the liver is not homogenously involved, it is possible for features of all four stages to occur in a single biopsy, leading to an over- or underestimation. In summary, diagnosis is based on three criteria: 1. cholestatic biochemical pattern, with elevated gGT, AP, and/or bilirubin 2. presence of AMA-M2 autoantibodies 3. features of PBC on liver histology. Two of three criteria are sufficient for the diagnosis of PBC.

9.3.2 Associated diseases PBC can be associated with osteoporosis due to malabsorption of both vitamin D and calcium in the later stages of the disease. Serum levels of vitamin A and E may then decrease as well, since cholestatic liver diseases, such as PBC, can lead to insufficient secretion of bile acids, which are required for proper absorption of dietary lipids and fat-soluble vitamins. The “sicca syndrome” is most frequently associated in up to 66% of PBC patients. Among patients with PBC, extrahepatic autoimmune diseases have been reported in up to 84% of patients (42). Another population-based cohort study reported at least one associated autoimmune disease in 53% of the PBC patients, with a higher risk in AMA-negative patients (79% vs. 49%) (43). uTab. 9.5 shows the most commonly associated immune-mediated diseases.

Tab. 9.5: Immune-mediated diseases associated with PBC Diseases associated with PBC Sjo¨gren’s syndrome (sicca syndrome) Scleroderma Raynaud’s disease CREST syndrome (calcinosis cutis, Raynaud’s phenomenon, esophagitis, sclerodactyly, teleangiectasias) Systemic lupus erythematosus Rheumatoid arthritis Mixed connective tissue disease Polymyositis, dermatomyositis, lichen planus, pemphigoid, psoriasis Thrombocytopenic purpura, pernicious anemia Sarcoidosis Myasthenia gravis Hashimoto thyreoiditis, Graves’ disease Diabetes mellitus type I, Addison’s disease Celiac disease, Crohn’s disease, ulcerative colitis

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19. Czaja AJ and Freese DK. (2002) Diagnosis and treatment of autoimmune hepatitis. Hepatology 2002;36:479–497. 20. Denzer U, Arnoldy A, Kanzler S, Galle PR, Dienes HP, Lohse AW: Prospective randomized comparison of minilaparoscopy and percutaneous liver biopsy: diagnosis of cirrhosis and complications. J Clin Gastroenterol. 2007;41(1):103–110. 21. Lu¨th S, Kanzler S, Frenzel C, Kasper HU, Dienes HP, Schramm C, et al.: Characteristics and long-term prognosis of the autoimmune hepatitis/primary sclerosing cholangitis overlap syndrome. J Clin Gastroenterol. 2009;43(1):75–80. 22. Lohse AW, zum Buschenfelde KH, Franz B, Kanzler S, Gerken G, Dienes HP: Characterization of the overlap syndrome of primary biliary cirrhosis (PBC) and autoimmune hepatitis: evidence for it being a hepatitic form of PBC in genetically susceptible individuals. Hepatology 1999;29:1078–1084. 23. Norris S, Kondeatis E, Collins R et al.: Mapping MHC-encoded susceptibility and resistance in primary sclerosing cholangitis: the role of MICA polymorphism. Gastroenterology 2001;120:1475–1482. 24. Tischendorf JJW, Hecker H, Kru¨ger M et al.: Characterization, outcome, and prognosis in 273 patients with primary sclerosing cholangitis: a single center study. Am J Gastroenterol 2007;102:107–114. 25. Rudolph G, Gotthardt D, Kloters-Plachky P et al.: Influence of dominant bile duct stenoses and biliary infections on outcome in primary sclerosing cholangitis. J Hepatol 2009;51:149–155. 26. Broome U, Olsson R, Loof L et al.: Natural history and prognostic factors in 305 swedish patients with primary sclerosing cholangitis. GUT 1996;38:610–615. 27. Karlsen TE, Schrumpf E, Boberg KM: Update on primary sclerosing cholangitis. Digestive and Liver Disease 2010;42:390–400. 28. EASL clinical practice guidelines: management of cholestatic liver disease. J Hepatol 2009;51:237–267. 29. Chapman RW, Fevery J, Kalloo AN et al.: AASLD guidelines: diagnosis and management of primary sclerosing cholangitis (PSC). Hepatology 2010;51:660–678. 30. Terjung B, Herzog V, Worman HJ, et al.: Atypical antineutrophilic cytoplasmic antibodies with perinuclear fluorescence in chronic inflammatory bowel disease and hepatobiliary disorders colocalize with nuclear lamina proteins. Hepatology 1998; 28:332–340. 31. Ashok KS: Role of MRCP versus ERCP in bile duct cholanciocarcinoma and benign stricture. Biomed Imaging Interv J 2007;3:e12–e545. 32. Kim JY, Kim MH, Lee TY et al.: Clinical role of F-FDG PET-CT in suspected and potentially operable cholangiocarcinoma: a prospective study compared with conventional imaging. Am J Gastroenterol 2008;103:1145–1151. 33. Tischendorf JJ, Kru¨ger M, Trautwein C et al.: Cholangioscopic characterization of bile duct stenoses in PSC. Endoscopy 2006;38:665–669. 34. Ghazale A, Chari ST, Zhang L, Smyrk TC, Takahashi N, Levy MJ: Immunoglobulin G4associated cholangitis: clinical profile and response to therapy. Gastroenterology. 2008; 134:706–715. 35. Hohenester S, Oude-Elferink RPJ, Beuers U: Primary biliary cirrhosis. Semin Immunpathol 2009;31:283–307. 36. Prince M et al.: Survival and symptom progression in a geographically based cohort of patients with primary biliary cirrhosis: follow-up for up to 28 years. Gastroenteroloy 2002;123:1044–1051. 37. Prince M et al.: Asymptomatic primary biliary cirrhosis: clinical features, prognosis, and symptom progression in a large population based cohort. GUT 2004;53:865–870.

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10 Autoimmunity in diabetes mellitus Peter Achenbach and Werner A. Scherbaum

10.1 General aspects and clinical features Type 1 diabetes (T1D) is a chronic disease caused by insulin deficiency that requires lifelong insulin therapy. Inadequate metabolic control places affected individuals at increased risk for serious acute or late-stage health problems. Intensive insulin therapy offers today’s most effective strategy to control hyperglycemia in patients with T1D, and maintenance of near-normal blood glucose control can reduce late diabetic complications (1). T1D is caused by an infiltration of the pancreatic islets with mononuclear cells, especially macrophages and autoreactive lymphocytes (insulitis), leading to a selective destruction of beta cells (2,3,4) and subsequently to insulin deficiency. The rate of destruction varies between individuals. Sudden onset and rapidly progressive T1D is commonly observed in children (5,6) but may also occur in adults (7), whereas a slowly progressive form generally occurs in adults and is referred to as latent autoimmune diabetes in adults (LADA) (8,9,10). This latter form may include an estimated 5% to 10% of patients diagnosed with type 2 diabetes (T2D) (11). The clinical presentation at diagnosis of T1D also varies. Some patients, particularly children and adolescents, may present with acute symptoms and ketoacidosis as the first manifestation of the disease. At this stage of disease there is little or no insulin secretion, as manifested by low levels of plasma C-peptide, and patients must be treated with exogenous insulin for survival. Others, particularly adults, may retain residual beta-cell function for many years. Although patients are usually not obese when they present with T1D, the presence of obesity is not incompatible with the diagnosis. Patients with T1D may also have other autoimmune disorders, such as Graves’ disease, Hashimoto’s thyroiditis, celiac disease, or Addison’s disease. T1D is a common feature in autoimmune polyendocrine syndrome type 2, but it is also rarely seen in children with autoimmune polyendocrine syndrome type 1 (12).

10.2 How does type 1 diabetes develop? – the autoimmune pathogenesis 10.2.1 The natural history of type 1 diabetes The model proposed by Atkinson and Eisenbarth for T1D development is based on clinical and experimental findings and has been in use for more than 20 years (uFig. 10.1) (13). It describes T1D as a chronic, progressive autoimmune disorder in which subjects at genetic risk experience an as yet undefined environmental insult that initiates the selective destruction of the insulin-producing beta cells, resulting in metabolic

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Fig. 10.1: Type 1 diabetes: a chronic autoimmune disease.

abnormalities such as impaired glucose tolerance and ultimately symptomatic hyperglycemia. The process of beta-cell destruction is accompanied by the appearance of cellular and humoral autoimmunity to beta-cell antigens, which may precede the development of clinical T1D by years. According to this model, distinct points can be defined at which preventive therapies may be implemented (uFig. 10.1). At stage 1, in the presence of genetic susceptibility, primary prevention may prevent the appearance of islet autoimmunity. At stage 2, triggering/immunizing event(s) for the initiation of islet autoimmunity occur. At stage 3, there is a progressive loss of glucose-stimulated insulin release; secondary prevention may then prevent the progression of islet autoimmunity to T1D. At stage 4, when the clinical manifestation of diabetes is seen, tertiary prevention may prevent the loss of residual beta-cell function.

10.2.2 Prospective studies from birth Over the past 20 years, several research groups have initiated prospective studies from birth in order to examine the development of islet autoimmunity and progression to diabetes, providing an opportunity to test this model in patients developing T1D. Findings from the German BABYDIAB study (14) and other projects have significantly contributed to the current understanding of the pathogenesis of childhood diabetes. It is now well known when islet autoimmunity first appears in life, which of the genetic factors influence its development, and which characteristics of islet autoantibodies are most associated with progression to T1D.

10.2.3 Major contributors to islet autoimmunity Three components are essential for the development of islet autoimmunity and T1D: genetic susceptibility, environmental factors, and defective immune regulation (15).

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10.2.3.1 Genetic susceptibility 10.2.3.1.1 Monogenetic forms of type 1 diabetes Two monogenetic forms of T1D can now be distinguished: 1. Autoimmune polyendocrine syndrome type 1 (APS-1), which is caused by mutations of the autoimmune regulator gene (AIRE) on chromosome 21 (16,17). Some 18% of patients with APS-1 develop T1D. 2. X-linked polyendocrinopathy, immune dysfunction, and diarrhoea (IPEX syndrome) (18), caused by mutations in the gene encoding the transcription factor forkhead box P3 (FOXP3) (19), which result in a lack of regulatory CD4+/CD25+ T lymphocytes and overwhelming autoimmunity. More than 90% of patients with IPEX syndrome develop T1D. Most of the affected children die in the first days of life or in infancy.

10.2.3.1.2 Idiopathic type 1 diabetes Genes determine susceptibility to T1D and can influence the appearance and progression of islet autoimmunity. All the major T1D susceptibility genes identified to date have in common a functional relationship to the immune system, including involvement in antigen presentation, antigen expression, immune stimulation, immune regulation, or signal transduction in immune cells (20). Whereas some genes contribute to immune dysregulation and breakdown of immune tolerance to islet autoantigens, others can be protective (21). Hence, a combination of various genes shapes T1D susceptibility in the majority of cases. It is important to appreciate that different combinations of genes contributing to an individual’s genetic susceptibility play a role in determining patterns of islet autoimmunity, associated diabetes risk, and/or speed of progression to disease. However, only a limited number of T1D-associated genes, mainly those in the HLA class II region on chromosome 6, are currently used for screening purposes.

10.2.3.2 Cell-mediated autoimmunity T1D is generally considered to be an immune cell–mediated disease. A cellular infiltrate (insulitis) (2,3) consisting predominantly of CD8+ and CD4+ T lymphocytes and variable numbers of B lymphocytes, macrophages, dendritic cells, and natural killer cells is present inside and around the pancreatic islets at and prior to diabetes onset (4). In humans, a pathogenic role for T cells is likely (22). Convincing evidence comes from transplant studies in identical twins, in which the transplant of pancreas segments from the unaffected twin to the diabetic twin resulted in a CD8+ T cell–predominant insulitis in the graft and the rapid loss of graft function (23). The role of B cells is less convincing. We described a child with Burton‘s syndrome (which lacks functional B lymphocytes), indicating that B cells are not strictly necessary for the development of T1D (24).

10.2.3.3 Autoantigens in type 1 diabetes The autoimmune response in T1D is specifically directed against molecular targets that are predominantly expressed in beta cells. Autoantibody and T-cell responses associated with T1D have been reported to a wide variety of molecules (uTab. 10.1).

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Tab. 10.1: Autoantigens in type 1 diabetes Antigen

Expression

Subcellular location Antibodies

T cells

Secretory Granule

Human, mouse

Human, mouse

Major T1D autoantigens Insulin

Islet-specific

GAD65

Neuroendocrine Synaptic-Like Microvesicle

Human

Human, mouse

IA-2 (ICA512)

Neuroendocrine Secretory Granule

Human

Human, mouse

IA-2β (phogrin)

Neuroendocrine Secretory Granule

Human

Human, mouse

ZnT8

Islet-specific

Secretory Granule

Human

Human

Human

Human, mouse

Minor or candidate T1D autoantigens Proinsulin

Islet-specific

Golgi Apparatus

preproIAPP

Islet-specific

Secretory Granule

Human, mouse

IGRP

Islet-specific

Endoplasmatic Reticulum

Mouse

HIP/PAP

Islet-specific

Secretory Granule

Mouse

Reg II

Islet-specific

Secretory Granule

Reg Iα

Islet-specific

Secretory Granule

Mouse

GAD67

Neuroendocrine Cytosol

Human

Human, mouse

ICA69

Neuroendocrine Golgi Apparatus

Human

Human, mouse

Carboxypeptidase H

Neuroendocrine Secretory Granule

Human, Mouse

Mouse

Glima 38

Neuroendocrine Secretory Granule

Human

Glycolipid GM2–1

Neuroendocrine Secretory Granule

Human

Ganglioside GT3

Neuroendocrine Cell Membrane

Human

Sulfatide

Neuroendocrine Secretory Granule

Human

Chromogranin A

Neuroendocrine Secretory Granule

Mouse

S100β

Neuroendocrine Cytosol

Human, mouse

Peripherin

Neuroendocrine Cytosol

Mouse

GLUT2

Widely

Cell Membrane

Human

DNA topoisomerase II

Widely

Nucleus

Human

SOX13 (ICA12)

Widely

Nucleus

Human

Human

Mouse

(Continued)

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Tab. 10.1: Continued Antigen

Expression

Subcellular location Antibodies

T cells

Jun-B

Widely

Nucleus

Human

Human

Imogen 38

Widely

Mitochondria

HSP60

Widely

Mitochondria

Human, mouse

Human Human, mouse

HSP70

Widely

Mitochondria, Cytosol, ER

Human

Human

HSP90

Widely

Cytosol

Human

AADC

Widely

Cytosol

Human

Whereas some of these antigens have been confirmed as major targets of the autoimmune process by many investigators (major T1D autoantigens), a number of others have been proposed but their relevance for the disease remains uncertain (minor or candidate T1D autoantigens). Interestingly, many of the autoantigens identified are related to cells of neuroendocrine origin with highly developed and regulated secretory mechanisms, and all of the major T1D autoantigens are related to the secretory apparatus. The best-studied major autoantigens in T1D are insulin and proinsulin, the 65-kDa isoform of glutamate decarboxylase (GAD65), and the protein tyrosine phosphatase (PTP)-related molecules IA-2 (ICA512) and IA-2β (phogrin). (Pro)insulin is present within beta-cell secretory granules, and IA-2 and IA-2β are transmembrane proteins in these granules, whereas GAD65 is a membrane-associated protein of beta-cell synaptic-like microvesicles. Most recently, another transmembrane protein of betacell secretory granules, zinc transporter-8 (ZnT8), has been identified as an additional major autoantigen in human T1D. The latter is also of interest because it signifies that the range of antigen targets is not exhausted, further emphasizing the involvement of critical autoantigens in insulin secretion and suggesting that other molecules could be identified that may serve as new diagnostic markers, therapeutic targets, and/or candidates for antigen-specific intervention/vaccination studies to prevent T1D.

10.3 Islet autoantibodies Autoantibodies to islet beta-cell antigens (islet autoantibodies) are a hallmark of T1D (15). They precede diabetes onset in more than 95% of children who develop disease and are frequent in insulin-requiring diabetes in adults. Although they are not considered effectors of islet beta-cell damage, they are established markers in the clinical classification of diabetes, prediction of the need for insulin treatment, identification of individuals at risk for developing T1D, and as endpoints in observational studies. Circulating islet autoantibodies are present in sera from new-onset T1D patients as well as prior to the clinical onset of disease, signaling an active and disease-specific B lymphocyte response. They can be very reproducibly detected and are excellent markers for identifying early immunization to beta-cell antigens, to follow progression to diabetes, and to identify individuals who are likely to develop T1D. The autoantibody

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profiles differ between individuals, have distinct features with respect to the age of first autoantibody appearance, and change on follow-up. The currently best-validated and most widely used predictive markers for T1D are autoantibodies directed against the biochemically defined target antigens insulin, GAD65, IA-2, and ZnT8. Apart from these, other autoantibodies have been detected in T1D, but their relevance as to the pathogenesis of the disease has not been confirmed in large studies or the target molecule has not been identified, so that their measurement remains too cumbersome for large-scale studies. First-degree relatives of patients with T1D are at increased risk, but in 90% of the cases affected individuals do not have a close relative with T1D. Population screening, in schoolchildren, for example (25), is required to identify individuals at risk, either by antibody testing or by a combination of genetic and antibody screening.

10.3.1 Major type 1 diabetes autoantibodies 10.3.1.1 Cytoplasmic islet-cell autoantibodies (ICAs) In 1974, Gian-Franco Bottazzo, A. Florin-Christensen, and Deborah Doniach first identified islet cell autoantibodies in T1D patients who had polyendocrine autoimmune deficiencies associated with organ-specific autoimmunity (26). These antibodies were detected by indirect immunofluorescence testing on sections of freshly frozen human pancreas (27). Complement-fixing islet-cell autoantibodies (ICAs) were found to be associated with active beta-cell damage (28). ICAs were subsequently found at a high frequency in children with newly diagnosed T1D and it was soon recognized that these antibodies are often present in the serum from relatives of patients with T1D, thus indicating a long prediabetic period with an autoimmune serology (29). Studies in identical twins and triplets of patients with T1D showed that the natural course of autoimmune T1D is very slow (30). ICAs are now quantified in Juvenile Diabetes Foundation Units (31). ICA levels are difficult to standardize because both the source of the pancreas as well as the antibodies are heterogeneous between individuals with respect to their molecular targets. ICA testing is therefore being superseded by testing for autoantibodies against biochemically defined islet antigens.

10.3.1.2 Insulin autoantibodies Although it had long been recognized that treatment with exogenous forms of insulin could induce antibodies directed against insulin peptides, in 1983 Jerry Palmer first described insulin autoantibodies (IAAs) in T1D patients before they received insulin therapy (32). Insulin and proinsulin are early target autoantigens of autoantibodies, are frequent (>70%) in childhood diabetes and are less prominent markers of T1D occurring after adolescence. IAAs (also called circulating insulin autoantibodies, or CIAAs) are usually the first autoantibodies to appear in young children who develop T1D (33) and can persist for many years before the clinical onset of T1D. The first occurrence of IAAs is marked at around 1 to 2 years of age, and there are distinct immunization patterns with respect to the affinity and epitope specificities of IAAs (34). The latter findings are highly relevant to pathogenesis and for distinguishing IAA-positive subjects who progress to diabetes from those who do not. In general the high-affinity IAAs are more predictive of T1D and share certain characteristics, including appearance

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at a young age, association with HLA DRB1*04, subsequent progression to multiple autoantibody positivity, binding to human insulin A chain residues 8 to 13, and binding to proinsulin.

10.3.1.3 GAD autoantibodies The next major islet cell autoantigen to be identified, in 1990, was a 65-kDa isoform of glutamate decarboxylase (GAD65) (35). Autoantibodies to GAD (GADAs) are frequent (>70%) in T1D, seen at all ages; they are the typical marker of autoimmune diabetes in adults (36). They are also detected in neurologic disorders, especially in stiff-man syndrome and in conditions unrelated to diabetes (37,38). Our group managed to generate human monoclonal islet cell autoantibodies (MICAs) shown to be directed to GAD (39). This enabled us to look into the structure of the autoantigen and to study the epitope specificity of GAD65 in T1D. Autoantibodies to GAD65 in patients with stiff-man syndrome also bind linear epitopes on the GAD molecule and can thus be detected by Western blotting. By contrast, autoantibodies in patients with T1D recognize conformational epitopes on the GAD65 molecule. Critical epitope clusters were identified that appear early in the GADA response (40). The affinity and epitope specificity of the antibody response was shown to predict progression in the disease process (41,42). In the early stages of T1D-associated GAD65 autoimmunity, the epitopes recognized by GADAs are primarily in the middle region of the protein, but later they may include regions at the N-terminus. Antibodies to GAD65 have sequence homology with the coxsackie B4–2c protein and with heat–shock protein-60 (43). GADAs are particularly important in the diagnosis of LADA patients.

10.3.1.4 IA-2 and IA-2β autoantibodies In 1995, Rabin and colleagues recognized that islet-cell antigen 512 (ICA512) is a diabetes-specific islet autoantigen related to protein tyrosine phosphatases (44). Two tryptic digest fragments of islet antigens were charcterized from individuals with T1D. One, a 40-kDa fragment from the intracellular portion of a tyrosine phosphatase-like protein (PTPRN gene), is now referred to as IA-2ic or ICA512ic. Autoantibodies to IA-2 (IA-2As) are almost always detected together with other islet autoantibodies and are therefore very specific for T1D (45). The other 37-kDa tryptic fragment was identified as the IA-2-related protein IA-2β or phogrin (46). Since almost all autoantibodies that react with IA-2β also react with IA-2, clinical laboratories typically do not use IA-2β autoantibodies as a first-line test. However, these antibodies may be useful for identifying individuals at high risk of disease progression. We and others have defined critical epitope regions/residues for IA-2A and IA-2β antibodies (47,48), and their hierarchy in pre-T1D was described. For example, subreactivity to the IA-2β protein is strongly associated with progression to diabetes within 5 years (49).

10.3.1.5 ZnT8 autoantibodies Of growing interest for autoimmune diabetes testing are autoantibodies to the beta cell−specific zinc transporter-8 (ZnT8), which were discovered in 2007 by screening for highly expressed islet beta cell−specific molecules (50). ZnT8 is one of a large family of zinc transporters that is associated with the membrane of secretory granules of

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islet beta cells; the zinc within these granules forms a complex with insulin to develop storage crystals (51). Autoantibodies to ZnT8 (ZnT8As) are found in about 70% of patients with T1D and improve the prediction of T1D. A principal epitope targeted by ZnT8As is influenced by the single amino acid at position 325 encoded as arginine, tryptophan, or glutamine by different polymorphic variants of the ZnT8-encoding gene SLC30A8 (52). It has been shown that autoimmunity against the COOH-terminal region of ZnT8 is a highly relevant prognostic feature of T1D development, and ZnT8Apositive children who are homozygous for either arginine or tryptophan at position 325 have the highest risk for developing T1D (53). Although testing of ICA on frozen sections of human pancreas tissue by immunofluorescence (27) has largely be replaced by radioimmunoassay or enzyme-linked immunosorbent assay (ELISA) detection of GADAs and IA-2As, it is still relevant in rare cases, since this method allows the detection of antibodies to as yet unidentified islet antigens, which can be helpful for diagnosis in few subjects who are negative for autoantibodies to the major islet antigens insulin, GAD, IA-2, and ZnT8.

10.3.2 Standardization of autoantibody measurement Exact and reproducible autoantibody measurement is a prerequisite for accurate prediction of T1D and diagnostic autoantibody testing in patients. The identification and molecular cloning of defined islet antigens has resulted in the rapid development of autoantibody assays, which have now been established in specialized laboratories worldwide. The Immunology of Diabetes Society (IDS) and the U.S. Centers for Disease Control and Prevention (CDC) established the Diabetes Antibody Standardization Program (DASP) in 2000 to improve comparability and to evaluate new autoantigens and test methodologies (31). The goal of the organization is to improve the detection and diagnosis of autoimmune diabetes by providing technical support, training, and information about the best methods; providing proficiency testing to evaluate laboratory performance; supporting the development of highly sensitive and specific measurement technologies; and developing reference materials. Since its inception, DASP has conducted six international workshop in which laboratories tests blinded samples from 50 patients with new-onset T1D and up to 100 controls. This format provides an evaluation of the sensitivity and specificity of each test and enables DASP to assess implementation of assay methods and to document any improvement in performance. DASP also provides an established platform for the rapid evaluation of new T1D markers and technologies by the wider research community (54). For GADAs and IA-2As, there is high concordance between laboratories. Radio-binding assays and some ELISAs can provide both high sensitivity and specificity. A World Health Organization (WHO) reference reagent is available allowing worldwide comparison of antibody levels (55), and standard protocols for the detection of GADA and IA-2As have been developed by the NIH/NIDDK Islet Autoantibody Assay Harmonization Program (56). However, concordance between laboratories is lower for IAAs (57). This is because of the requirement that assays be able to reproducibly detect low titers of IAAs. To date, only a few assays, all radio-binding assays, have sufficient sensitivity and specificity to be considered useful for measuring IAAs in preclinical T1D. Before using an islet autoantibody assay for T1D risk assessment and diagnostic purposes, its performance in the IDS/CDC-based international workshops should be ascertained.

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10.3.3 The use of autoantibodies for diabetes classification The measurement of islet autoantibodies to help distinguishing between autoimmune (T1D) and nonautoimmune (mainly T2D) diabetes has become increasingly important in recent years. Traditionally, clinicians have differentiated between T1D and T2D based on phenotypic characteristics, including age at onset, abruptness of onset of hyperglycemia, proneness to ketosis, degree of obesity, prevalence of autoimmune disease, and the need for insulin replacement therapy. However, clear discrimination between type 1 and type 2 diabetes on the basis of clinical parameters is often difficult, since some patients with T1D may be overweight; they may also have a subacute onset of symptoms and may initially be well controlled with oral antidiabetic drugs alone.

10.3.3.1 Latent-onset autoimmune diabetes in adults (LADA) A portion of patients who initially present with the clinical features of non−insulin dependent diabetes may progress to insulin dependency within several months or only a few years. These patients are usually nonobese adults between 35 and 50 years of age and are characterized by the presence of T1D-specific autoantibodies in the serum. Patients with LADA have cytoplasmic islet cell antibodies, specifically autoantibodies to GAD and less frequently also to IA-2 (58,59). They have low c-peptide levels (60) that tend to further decrease within 3 years (61,62). Patients in the LADA category present clinically without ketoacidosis. LADA is often misdiagnosed as T2D. Ten percent of patients on the UKPDS trial who were clinically diagnosed as having T2D tested positive for GADA (11).

10.3.3.2 GADA in women with gestational diabetes Some 17% of women with insulin-requiring gestational diabetes (white class B) in the German Gestational Diabetes Study had GADA at delivery and developed insulindependent diabetes postpartum (63). Therefore measurement of islet autoantibodies at diabetes diagnosis is useful in overweight children, nonobese adults with non−insulin dependent diabetes, and in women with gestational diabetes who require insulin during pregnancy. The most sensitive antibodies to be measured for purpose of distinguishing between T1D and T2D are GADAs in adults and a combination of GADAs, IA-2As, and IAAs in young children. More than 80% of patients with newly diagnosed T1D have GADAs, 70% have IA-2As, and around 60% have IAAs.

10.4 Type 1 diabetes risk screening Assessment of T1D risk and prediction of disease development remain primarily research tools, useful for identifying subjects suitable for recruitment into intervention trials aiming to prevent the clinical onset of T1D. In recent years, the characteristics of the stages of pre-T1D have been studied in different cohorts of individuals at risk, leading to the identification of disease-associated biomarkers that can now be applied to risk assessment and disease prediction. These studies have also demonstrated that progression to disease is not uniform and that, in order to optimize risk assessment,

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demographic, genetic, immune, and metabolic predictive markers should be combined. Current strategies enable family members of patients with T1D to be stratified into the majority who have levels of risk equivalent to those of the background population and a small proportion at high risk for rapid progression to disease. Recruitment into future trials can therefore be based on the careful selection of participants who are best suited for the individual therapeutic approaches. A number of different screening tools are available, but both their value and acceptable financial and other costs will depend on the purpose of risk assessment. Primary prevention trials aim at preventing the development of T1D by intervening at the earliest stage of pathogenesis, before the onset of islet autoimmunity. Primary prevention is therefore applicable to islet autoantibody-negative young children who are at high genetic risk of T1D. In contrast, secondary prevention trials apply intervention to nondiabetic individuals who have already developed islet autoimmunity with the aim of preventing progression to clinical disease. Islet autoantibody−positive relatives of people with T1D are currently the cohort of choice for recruitment into secondary prevention trials.

10.4.1 Screening for islet autoimmunity 10.4.1.1 First screening step Individuals with evidence of islet autoimmunity may be suitable for inclusion into secondary prevention trials. Selection of participants for such trials therefore requires screening for immune markers, which in T1D is currently synonymous with measurement of islet autoantibodies in peripheral blood.

10.4.1.1.1 Islet autoantibodies in relatives Much of our current understanding of islet autoantibodies and their role in prediction has derived from prospective studies in individuals with an increased genetic susceptibility, such as relatives of patients with T1D. The prevalence of islet autoantibodies in relatives is 5% to 10%, depending upon which antibodies are measured. The largest screening in relatives has been undertaken as part of the Diabetes Prevention Trial Type 1 (DPT-1) in North America (64). In a DPT-1 substudy, samples from 17,207 of the 71,148 first-degree relatives tested for ICAs, GADAs, and IA-2As were also tested for IAAs by microassay. At least one of the four autoantibodies (above the 99th percentile) was found in 8.2% of relatives tested and more than one autoantibody in 2.3% (65). Although islet autoantibodies are closely associated with future disease, not all subjects with such autoantibodies will develop T1D. Substantial efforts have therefore been made to identify disease-specific characteristics of autoantibodies and other markers that will help distinguish which islet autoantibody−positive relatives will and will not develop T1D, and, if so, when this is likely to occur.

10.4.1.1.2 Screening in young children from affected families It has been demonstrated that IAAs are almost always the first autoantibodies to appear in young children who subsequently progress to T1D and that the typical natural history of T1D in children is the appearance of IAAs followed by relatively early/rapid spreading to other islet autoantibodies and eventually the development of diabetes (15). T1D

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risk screening in children, adolescents, and young adults (up to around 25 years of age) should therefore include all T1D-associated major autoantibodies (i.e. IAAs, GADAs, IA-2As, and ZnT8As). The prevalence of IAAs is largely age-dependent, and around 90% of children diagnosed before the age of 5 years are IAA-positive. It is unclear whether the lower frequency of IAAs found after adolescence is due to the fact that these patients never developed insulin autoimmunity or due to the loss of IAAs during the preclinical phase of the disease.

10.4.1.1.3 Screening in older relatives Screening in older relatives should take into consideration the frequencies of the individual antibodies in older T1D cases. The prevalence of IAAs decreases dramatically with increasing age; IAAs are therefore not particularly useful screening markers in the groups above 25 years of age. The prevalence of GADAs on the other hand, is relatively stable with age. IA-2As are slightly more prevalent in younger cases, whereas the prevalence of ZnT8As is directly correlated with the age of T1D onset. Therefore, adults (older than 25 years) should be screened for GADAs, IA-2As, and ZnT8As, with additional sequential testing for IAAs for further risk stratification.

10.4.1.1.4 Screening on a population level Islet autoantibody screening on a population level is possible, but the prevalence of positive results is only between 1% and 2%, with an infrequent finding of high-titer autoantibodies to multiple islet antigens. However, if present, these also denote an increased risk for the further progression to diabetes (66).

10.4.1.1.5 Screening in patients with endocrine autoimmune diseases T1D may develop as part of an autoimmune polyendocrine syndrome (APS) type 1 or APS type 2 (12). Patients with APS-1 have a dysregulated immunity and a number of potential associated autoimmune disorders including primary hypoparathyroidism, autoimmune thyroid disorders, autoimmune Addison’s disease, T1D, and others. APS-2 is often present in patients with autoimmune Addison’s disease, so that testing for islet autoantibodies is recommended to detect a prediabetic condition. The presence of autoantibodies to islet antigens in patients with single autoimmune thyroid disease and other autoimmune endocrine disorders is less common (67), but if such autoantibodies are present, an increased risk for the future development of T1D is highlighted (68).

10.4.1.2 Second screening step Once islet antibodies are detected, repeated testing is valuable in order to determine whether the antibodies persist and more antibodies have developed. Both features are relevant for the prognosis. Only persistent islet autoantibodies signal an active autoimmune process relevant to diabetes development. Single positive test results or transient autoantibody appearance is not associated with progression to T1D (69,70). IAAs are least likely to persist among the major islet autoantibodies, and this is related to antibody titer.

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If islet autoantibodies have been confirmed as stable, markers of progression should be looked for. These may be immune and/or metabolic, may only develop during follow-up, and could potentially indicate relevant ”events” in the underlying pathogenesis. It is therefore important to monitor autoantibodies at appropriate time intervals, especially in young individuals in whom changes are usually more frequent and rapid (71).

10.4.1.2.1 Multiple islet autoantibodies The development of multiple islet autoantibodies is a critical step in pathogenesis and therefore a highly relevant marker of risk of progression. It has been known for almost two decades that detection of two or more islet autoantibodies is associated with a significantly higher T1D risk than a single autoantibody (72,73,74). Whereas T1D risk is less than 20% in relatives with just one islet autoantibody, it is around 35% within 5 years and 61% within 10 years in those with more than one autoantibody (49). Multiple islet autoantibody−positive subjects without a T1D family history also appear to have a high risk. However, the status ”multiple autoantibody−positive” depends on which markers are tested. For example, around two thirds of relatives found to be GADA-positive but IAA- and IA-2A−negative (i.e., previously defined as low risk) in fact had autoantibodies to the newly identified autoantigen ZnT8, moving them into the multiple antibody−positive category. It is therefore possible that further marker(s) will become available that can identify more advanced islet autoimmunity and higher risk among individuals who are currently categorized as single autoantibody−positive.

10.4.2 Markers of progressive autoimmune insulitis More recently, characteristics of islet autoantibodies have been shown to identify relatives whose islet autoimmunity will progress. The major discriminating characteristics are related to the target specificity, the maturity and magnitude of the response, and the age at autoantibody appearance. Maturity and magnitude are reflected by antibody affinity, titer, and number of different IgG subclasses and target epitopes on individual and combined islet antigens. We found that autoantibodies to IA-2 restricted to the IgG4 subclass are associated with protection from T1D (75).

10.4.2.1.1 Target specificity There appears to be a hierarchy of diabetes relevance in the autoantibody response against different antigenic targets within and between islet autoantigens. For example, whereas risk is relatively low in relatives with GADAs or IAAs alone (around 20% within 10 years), the presence of IA-2As alone is associated with a similar risk (around 50% within 10 years) to multiple non-IA-2-autoantibodies (ICAs, GADAs and/or IAAs) (uFig 10.2) (49). Among IA-2A-positive relatives, risk can be further stratified according to the presence or absence of autoantibodies to IA-2β (76). Also, IAAs without proinsulin reactivity are associated with low risk, whereas proinsulin-reactive IAAs are associated with very high risk of progression (34). For GADAs, the N-terminal GAD-restricted antibodies are associated with low/no risk of progression, whereas individuals with antibodies directed toward the middle and/or C-terminal of the antigen progress to disease (42,77).

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Fig. 10.2: Progression to diabetes: antigen target matters.

10.4.2.1.2 Maturity of antibodies Antibody affinity provides a measure of maturity of the immune response. In a typical antibody response, exposure to antigen in the presence of B-cell growth factors results in B-lymphocyte expansion and IgM antibody production. Sustained or repeated antigen exposure leads to a switch from IgM to IgG production, and selection of clones that produce antibodies of high affinity to the antigen. In T1D, high-affinity autoantibodies are associated with progression of islet autoimmunity and are therefore ”diabetesrelevant,” whereas low-affinity antibodies are unrelated to diabetes development. The German BABYDIAB study has identified affinity as a marker for T1D-relevant IAAs and GADAs (34,42). IAA affinity varied considerably between IAA-positive children, and those who developed high-affinity IAAs had persistent IAAs, developed multiple islet autoantibodies, and had a 50% risk of developing T1D within 6 years. In contrast, children with IAAs of lower affinity rarely progressed to multiple islet autoantibodies and did not develop T1D. Furthermore, high-affinity IAAs differed from lower-affinity IAAs in insulin-binding characteristics, suggesting distinct epitope recognition and, in contrast to the lower-affinity IAAs, the associated epitope was also expressed on the proinsulin molecule. Similar findings were obtained for GADAs in that singlehigh-affinity GADA−positive children progressed to multiple islet autoantibodies and T1D more frequently than children with low-affinity GADAs.

10.4.2.1.3 Magnitude of antibody response The magnitude of an autoantibody response is reflected by persistence, titer, affinity, and the breadth or range of autoantigen targets. Diabetes development has been associated with high-titer ICAs, IAAs, or IA-2As. High titer also determines other characteristics, such as breadth of response in terms of IgG subclass usage and epitope reactivity. As expected, high-titer responses are usually synonymous with multiple IgG subclass antibodies to multiple epitopes, although these features can also be independent indicators of disease risk in low-titer autoantibody-positive subjects. In an analysis of

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Fig. 10.3: Model indicating antibody markers for progression of autoimmunity to diabetes.

autoantibody-positive relatives followed for up to 15 years, the highest risks for T1D were associated with high-titer IAA and IA-2A responses, with the appearance of antibody subclasses IgG2, IgG3, and/or IgG4 of IAAs and IA-2As and antibodies to the IA-2−related molecule IA-2β (uFig. 10.3) (49). Using various combinations of these islet autoantibody characteristics, it was possible to stratify 5-year diabetes risk from less than 10% to around 90%. High-titer autoantibodies to multiple islet antigens rarely disappear completely prior to the onset of diabetes.

10.4.3 Age at autoantibody appearance Risk of developing T1D can be stratified on the basis of how early islet autoantibodies develop. The earlier in life the first autoantibody appears and the more antibodies are present, the higher the risk for rapid progression of islet autoimmunity and diabetes. In the German BABYDIAB cohort, 50% of children who already had multiple islet autoantibodies within the first year of life progressed to diabetes within 2 years of follow-up (78). Children who develop islet autoantibodies before age 2 years frequently have high-affinity IAAs and progress to multiple islet autoantibodies, whereas children who develop autoantibodies after age 2 years are less frequently IAA-positive, are often GADA-positive, and infrequently develop multiple islet autoantibodies. It is also evident from the UKPDS that antibody-positive patients with non–insulin dependent diabetes who are below age 45 progress to insulin dependency more rapidly than patients over age 45, even if they have the same antibody patterns and levels (11).

10.4.4 Metabolic markers of progression to T1D Metabolic markers are not primary screening tools in T1D risk assessment but can further refine risk assessment in autoantibody-positive individuals. First-phase insulin

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response (FPIR) in the intravenous glucose tolerance test (IVGTT) is often impaired prior to diabetes onset, and there have been attempts to determine the rate of progression to diabetes by combining islet autoantibody measurement and FPIR (30). Autoantibodypositive relatives with a low FPIR have a faster rate of progression or are closer to diabetes than those with normal FPIR. In the Diabetes Prevention Trial – Type 1 (DPT-1), T1D risk in ICA-positive, IAA-positive relatives with FPIR below the first percentile was around 60% within 5 years (79). In the Deutsche Nicotinamid Interventionsstudie (DENIS) (80) and the European Nicotinamide Diabetes Intervention Trial (ENDIT) (81), the overall 5-year risk in ICA-positive relatives with low FPIR was around 55%; but among relatives positive for five autoantibodies (ICA, GADA, IA-2A, IAA, IA-2βA), the risk associated with a low FPIR increased to greater than 90%, and impaired oral glucose tolerance identified those with the fastest progression to disease (>50% progression within 1 year) (76). Recent studies have also suggested that combining measures of insulin resistance and FPIR in islet autoantibody−positive relatives may further contribute to risk assessment. Although risk assessment is clearly improved by the addition of metabolic measurements and the IVGTT and OGTT can stage the preclinical phase of T1D, these tests are difficult to standardize. Low FPIR was associated with T1D-related autoantibody characteristics − such as high-titer ICAs, the presence of IAAs, and multiple islet autoantibodies in children aged 1 to 5 years in the DIPP study as well as in older relatives participating in ENDIT − indicating that some of the increased risk conferred by low FPIR can be attributed to autoantibody characteristics. In accord with this, accurate T1D risk stratification was achieved on the basis of autoantibody characteristics that included titer, subclasses, and/or epitopes alone, suggesting that FPIR may not need to be included in the inclusion criteria for recruitment for future trials. It has been shown that rituximab markedly and selectively suppresses IAAs, clearly blocking formation of these antibodies for more than 1 year in insulintreated patients (82). Studies in prediabetic non–insulin treated patients will be needed to evaluate the specific effects of rituximab on IAAs and eventual prevention of diabetes at this stage of the disease process. Intervention studies in LADA are a new option; since in this slowly progressive form of autoimmune diabetes, there is a chance for appropriate intervention before most of the beta cells are lost. An intervention study with early insulin therapy gave promising results (83). Our large European consortium ACTION LADA will now enable large-scale prevention trials with a variety of candidate substances that either modulate the autoimmune process or/and improve beta-cell regeneration.

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24. Martin S, Wolf-Eichbaum D, Duinkerken G, Scherbaum WA, Kolb H, Noordzij JG, et al.: Development of type 1 diabetes despite severe heriditary B-cell deficiency. N Engl J Med 2001;345:1036–1040 25. Boehm BO, Manfras B, Seissler J, Scho¨ffling K, Glu¨ck M, Holzberger G, et al.: Epidemiology and immunogenetic background of islet cell antibody–positive nondiabetic schoolchildren. Ulm-Frankfurt population study. Diabetes 1991;40:1435–1439. 26. Bottazzo GF, Florin-Christensen A, Doniach D: Islet-cell antibodies in diabetes mellitus with autoimmune polyendocrine deficiencies. Lancet 1974;2:1279–1283. 27. Scherbaum WA, Mirakian R, Pujol-Borrell R, Dean BM, Bottazzo GF: Indirect Immunofluorescence in the Study of Organ-Specific Autoimmune Diseases, 346–361. In: J.M. Polak, S. Van Noorden (eds). Immunochemistry. Practical Applications in Pathology and Biology. Wright, Bristol-London-Boston, 1983. 28. Bottazzo GF, Dean BM, Gorsuch AN, Cudworth AG, Doniach D: Complement-fixing islet-cell antibodies in Type 1 diabetes: possible monitors of active beta cell damage. Lancet 1980;1:668–672. 29. Gorsuch AN, Spencer KM, Lister J, McNally JM, Dean BM, Bottazzo GF, et al.: Evidence for a long prediabetic period in type 1 (insulin-dependent) diabetes mellitus. Lancet 1981;2:1363–1365. 30. Srikanta S, Ganda OP, Eisenbarth GS, Soeldner JS: Islet-cell antibodies and beta-cell function in monocygotic triplets and twins initially discordant for type 1 diabetes mellitus. N Engl J Med 1983;308:322–325. 31. Bingley PJ, Bonifacio E, Mueller PW: Diabetes antibody standardization program: First assay proficiency evaluation. Diabetes 2003;52:1128–1136. 32. Palmer JP, Asplin CM, Ciemos P, Lyen K, Tatpati O, Raghu PK, et al.: Insulin antibosies in insulin-dependent diabetes before insulin treatment. Science 1983;222:1337–1339. 33. Ziegler AG, Schmid S, Huber D, Hummel M, Bonifacio E: Early infant feeding and risk of developing type 1 diabetes-associated autoantibodies. JAMA 2003;290:1721–1728. 34. Achenbach P, Koczwara K, Knopff A, Naserke H, Ziegler AG, Bonifacio E: Mature highaffinity immune responses to (pro)insulin anticipitate the autoimmune cascade that leads to type 1 diabetes. J Clin Invest 2004;114:589–597. 35. Baekkeskov S, Aanstoot AJ, Christgau S, Reetz A, Solimena M, Cascalho M, et al.: Identification of the 64K autoantigen in insulin-dependent diabetes as the GABA-synthesizing enzyme glutamate acid decarboxylase. Nature 1990;347:151–156. 36. Seissler J, Amann J, Mauch L, Haubruck H, Wolfahrt S, Bieg S, et al.: Prevalence of autoantibodies to the Mr 65,000 and Mr 67,000 isoforms of glutamate decarboxylase (GAD) in insulin-dependent diabetes mellitus. J Clin Invest 1993;92:1394–1399. 37. Solimena M, Folli F, Aparisi R, Pozza G, De Camilli G: Autoantibodies to GABAergic neurons and pancreatic beta cells in Stiff-Man Syndrome. N Engl J Med 1990;322:1555–1560. 38. Meinck HM, Maile S, Morgenthaler NG, Seissler J, Faber L, Butler M, et al.: Antibodies against glutamic acid decarboxylase (GAD): prevalence in neurological diseases. J. Neurol. Neurosurg. Psychiatry 2001;71:100–103. 39. Richter W, Endl J, Eiermann TH, Brandt M, Kientsch-Engel R, Thivolet C, et al.: Human monoclonal islet cell antibodies from a patient with insulin-dependent diabetes mellitus reveal glutamate decarboxylase as the target antigen. Proc Natl Acad Sci U S A 1992; 89:8467–8471. 40. Bonifacio E, Lampasona V, Bernasconi L, Ziegler AG: Maturation of the humoral autoimmune response to epitopes of GAD in preclinical childhood type 1 diabetes. Diabetes 2000;49:202–208. 41. Richter W, Seissler J, Northemann W, Wolfahrt S, Meinck HM, Scherbaum WA: Cytoplasmic islet cell antibodies recognize distinct islet antigens in IDDM but not in stiff man syndrome. Diabetes 1993;42:1642–1648.

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42. Mayr A, Schlosser M, Grober N, Kenk H, Ziegler AG, Bonifacio E, et al.: GAD autoantibody affinity and epitope specificity identify distinct immunization profiles in children at risk for type 1 diabetes. Diabetes 2007;56:1527–1533. 43. Richter W, Mertens T, Schoel B, Muir P, Ritzkowsky A, Scherbaum WA, et al.: Sequence homology of the diabetes-associated autoantigen glutamate decarboxylase with coxsackie B4–2C protein and heat shock protein 60 mediates no molecular mimicry of autoantibodies. J Exp Med 1994;180:721–726. 44. Rabin DU, Pleasic SM, Shapiro JA, Yoo-Warren H, Oles J, Hicks JM, et al.: Islet cell antigen 512 is a diabetes-specific islet autoantigen related to protein tyrosine phosphatises. J Immunol 1994;152:3183–3188. 45. Christie M, Genovese S, Cassidy D et al.: Antibodies to islet 37k antigen, but not to glutamate decarboxylase, discriminate rapid progression to Type 1 diabetes in endocrine autoimmunity. Diabetes 1994;43:1254–1259. 46. Lu J, Li Q, Xie H, Chen ZJ, Borovitskaya AE, Maclaren NK, et al.: Identification of a second transmembrane protein tyrosine phosphatise, IA-2beta, as an autoantigen in insulindependent diabetes mellitus: Precursor of the 37-kDa tryptic fragment. Proc. Natl. Acad. Sci. USA. 1996;93:2307–2311. 47. Seissler J, Schott M, Morgenthaler NG, Scherbaum WA: Mapping of novel autoreactive epitopes of the diabetes-associated autoantigen IA-2. Clin Exp Immunol 2000;122:157–163. 48. Bonifacio E, Lampasona V, Bingley PJ. IA-2 (islet cell antigen 512) is the primary target of humoral autoimmunity against type 1 diabetes-associated tyrosine phosphatase autoantigens. J Immunol 1998;161:2648–2654. 49. Achenbach P, Warncke K, Reiter J et al.: Stratification of type 1 diabetes risk on the basis of iset auoantibody characteristics. Diabetes 2004;53:384–392. 50. Wenzlau JM, Juhl K, Yu L et al.: The cation efflux transporter ZnT8 (Slc30A8) is a major autoantigen in human type 1 diabetes. Proc Natl Acad Sci U S A. 2007;104:17040–17045. 51. Chimienti F, Devergnas S, Favier A, Seve M: Indentification oa´ nd cloning of a betacell-specific zink transporter, ZnT-8, localized into insulin secretory granules. Diabetes 2004;53:2330–2337. 52. Wenzlau JM, Liu Y, Yu L et al.: A common nonsynonymous single nucleotide polymorphism in the SLC30A8 gene determines ZnT8 autoantibody specificity in type 1 diabetes. Diabetes 2008;57:2693–2697. 53. Achenbach P, Lampasona V, Landherr U, Koczwara K, Krause S, Grallert H, et al.: Autoantibodies to zinc transporter 8 and SLC30A8 genotype stratify type 1 diabetes risk. Diabetologia 2009;52:1881–1888. 54. Achenbach P, Schlosser M, Williams AJ, Yu L, Mueller PW, Bingley PJet al.: Combined testing of antibody titre and affinity improves insulin autoantibody measurement. Diabetes Antibody Standardization Program. Clin Immunol 2007;122:85–90. 55. To¨rn C, Mueller PW, Schlosser M, Bonifacio E, Bingley PJ: Diabetes Antibody Standardization Program: evaluation of assays for autoantibodies to glutamate acid decarboxylase and islet antigen-2. Diabetologia 2008;51:846–852. 56. Bonifacio E, Yu L, Williams AK, Eisenbarth GS, Bingley PJ, Marcovina SM, et al.: Harmonization of glutamic acid decarboxylase and islet antigen-2 autoantibody assays for National Institute of Diabetes and Digestive and Kidney Diaseases Consortia. JCEM 2010;95:3360–3367. 57. Schlosser M, Mueller PW, To¨rn C, Bonifacio E, Bingley PJ: Diabetes antibody standardization program: evaluation of assays for insulin autoantibodies. Diabetologia 2010; 53:2811–2820. 58. Tuomi T, Groop LC, Zimmet PZ, Rowley MJ, Knowles W, Mackay IR: Antibodies to glutamic acid decarboxylase reveal latent autoimmune diabetes mellitus in adults with a non-insulin-dependent onset of disease. Diabetes 1993;42:359–362

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75. Seissler J, Eikamp K, Schott M, Scherbaum WA: IA-2 autoantibodies restricted to the IgG4 subclass are associated with protection from type 1 diabetes. Horm Metab Res 2002;34:186–191. 76. Achenbach P, Bonifacio E, Williams AJ, Ziegler AG, Gale EA, Bingley PJ and the ENDIT Group: Autoantibodies to IA-2beta improve diabetes risk assessment in high-risk relatives. Diabetologia 2008;51:488–492. 77. Tree TI, Morgenthaler NG, Duhindan N, Hicks KE, Madec AM, Scherbaum WAet al.: Two amino acids in glutamic acid decarboxylase act in concert for maintenance of conformational determinants recognised by Type I diabetic autoantibodies. Diabetologia 2000;43:881–889. 78. Hummel M, Bonifacio E, Schmid S, Walter M, Knopff A, Ziegler AG: Brief communication: early appearance of islet autoantibodies predicts childhood type 1 diabetes in offspring of diabetic parents. Ann Intern Med 2004;140:882–886. 79. Vehik K, Cuthbertson D, Ruhlig H, Schatz DA, Peakman M, Krischer JP, for the DPT-1 and TrialNet Study Groups: Long-term outcome of individuals treated with oral insulin: Diabetes Prevention Trial-Type 1 (DPT-1) oral insulin trial. Diabetes Care 2011; 34:1585–1590. 80. Lampeter EF, Klinghammer A, Scherbaum WA, Heinze E, Haastert B, Giani G, Kolb H: The Deutsche Nicotinamide Intervention Study: an attempt to prevent type 1 diabetes. DENIS Group. Diabetes 1998;47:980–984. 81. Bingley PJ, Gale EA: Progression to type 1 diabetes in islet cell antibody-positive relatives in the European Nicotinamide Diabetes Intervention Trial: the role of additional immune, genetic and metabolic markers of risk. Diabetologia 2006;49:881–890. 82. Yu L, Herold K, Krause-Steiunrauf H, McGee PL, Bundy B, Pugliese A, et al.: Rituximab selectively suppresses specific islet antibodies. Diabetes 2011;60:2560–2565. http://creative commons.org/license/content/early/by-nc-nd/3.0/ 83. Maruyama T, Tanaka S, Shimada A, Funae O, Kasuga A, Kanatsuka A, et al.: Insulin intervention in slowly progressive insulin-dependent (type 1) diabetes mellitus. JCEM 2008; 93:2115–2121.

11 Autoimmune thyroid diseases Matthias Schott, Werner A. Scherbaum and Jochen Seissler

11.1 General aspects and clinical features Autoimmune thyroid diseases (AITDs) encompass a number of conditions that have in common cellular and humoral immune responses targeted at the thyroid gland. The classic AITDs include Graves’ disease (GD) and Hashimoto’s thyroiditis (HT), both of which involve infiltration of the thyroid by T and B cells reactive with thyroid antigens and the production of thyroid autoantibodies, with the resultant clinical manifestations (hyperthyroidism in GD and hypothyroidism in HT). Additional variants of AITD include postpartum thyroiditis, drug-induced thyroiditis (e.g., caused by amiodarone and interferon- alpha, thyroiditis accompanying the polyglandular autoimmune syndromes, and the presence of thyroid antibodies [Tabs] with no apparent clinical disease). Although the exact etiology of the immune response to the thyroid remains unknown, there is solid evidence of a major genetic influence on the development of AITDs. Therefore the current paradigm is that AITDs are complex diseases in which susceptibility genes and environmental triggers act in concert to initiate an autoimmune response to the thyroid.

11.2 Etiology Autoimmune thyroiditis is the most common autoimmune disease in humans. The prevalence ranged between 10% and 15%. There are two forms: hypertrophic and atrophic. The hypertrophic form, which is more common in children and young adults, may lead to the atrophic form during follow-up. The etiology of AITDs, including HT, is as yet unknown. From the pathophysiologic point of view, there is a lymphatic infiltration into the thyroid gland resulting in the destruction of thyroid follicles. The major autoantigen is thyroid peroxidase (TPO). The large thyroglobulin molecule may not present as the major autoantigen. GD is the only autoimmune disease of humans without signs of destruction of the target organ. The major phenomenon in GD is the generation of TSH receptor autoantibodies (TRAbs), which bind to the TSH receptor, resulting in their activation and the induction of hyperthyroidism.

11.3 Clinical features Clinical symptoms of HT are highly variable. Goiter, the hallmark of classic HT, usually develops gradually and may be found during routine examination. Patients with HT may initially develop hyperthyroidism, a high heart rate, weight loss, sweating, diarrhea, dizziness, and so on. Most of the patients, however, remain in a euthyroid state. Patients with hypothyroidism are tired, complain of obstipation, and may have signs of a myxedema as well as other symptoms.

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11.4 Workflow for the diagnosis of autoimmune thyroid disease Based on clinical symptoms, ultrasonography is a good diagnostic tool. Patients with autoimmune thyroiditis typically show a hypoechogenic appearance of the thyroid parenchyma. This is also true for patients with GD. In HT, however, the blood flow is normal or decreased (rarely initially increased), whereas GD patients typically show a largely increased blood flow. In thyrotoxicosis, this may even result in a thyroid storm. Scinitgraphy, which is rarely indicated, show an enlarged Tc uptake. A very sensitive parameter to investigate the function of the thyroid gland is the thyreotropin (TSH). Downregulated (4.5 μ/mL) may indicate a hypothyroidism. Based on the TSH level, free T3 and free T4 should be measured. Because of the low costs of measuring all three values, many clinicians directly measure all three values. By using this approach, “low T3 syndrome” can also be diagnosed.The diagnosis of HT or GD is confirmed by the finding of thyroid antibodies, usually by high titers of TRAbs in GD and TPO-Abs and/or Tg-Abs in HT. Since TPO-Abs may also be present in GD (in 60%–80% of cases) as well as low-titer TRAbs in HT (in less than 5% of cases), thyroid antibodies are discussed separately here.

11.4.1 TSH receptor autoantibodies In 1956, Adams and Purves described a thyroid-stimulating factor in serum samples of patients with hyperthyroidism (1), later identified to be autoantibodies (TRAbs) to the thyroid stimulating hormone (TSH) receptor (TSHR) (2). Today, very sensitive assays to detect TRAbs are commercially available. TRAbs are found principally in all patients with GD and are responsible for hyperthyroidism due to specific binding to TSHR (3). The widespread TRAb measurement has identified GD as the most prevalent overt autoimmune disease in the United States (4), affecting 1.2% of the population or 2.4% of females, respectively. In contrast, rheumatoid arthritis and overt autoimmune hypothyroidism are prevalent in about 1% and autoimmune diabetes in about 0.2% of the population.

11.4.2 TSH receptor immunobiology Over the past two decades, many groups have focused on the interaction between TRAb and the TSHR and studied the processing of the TSHR protein on the cell surface. Because of the brevity of this review, it is not possible to summarize all data. This chapter focuses only on some of the most interesting aspects. In general, it is now accepted that the single-chain TSHR is posttranslationally cleaved on the cell surface into TSHR-A (the N-terminal extracellular portion) and TSHR-B (membrane-bound) subunits, which remain linked by disulfide bonds. It has been proposed that the shed A subunit (rather than the holoreceptor) is the crucial autoantigen in the generation of thyroid-stimulating autoantibodies (TSAbs) (5). This follows earlier observations that TSAbs seem to recognize the N-terminal ectodomain of the receptor (essentially the A subunit) more efficiently when it is not linked to the serpentine portion 21 and 22. The role of the B subunit is less well understood. There is evidence that truncated TSHR constructs corresponding to B subunits have increased constitutive activity when expressed in nonthyroid cells (6,7,8), although this is still controversial (9).

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Even more controversial data exist on the binding of TSAbs and TBAbs. Initially, it was thought that TSAbs and TBAbs bind to the C-terminal and the N-terminal part, respectively, of the TSHR ectodomain. More recent data on the basis of monoclonal TSAbs and TBAbs, however, suggest that binding sites are in close proximity (10). In contrast to these data, it has also been shown that a component of the TSAb epitope is focused on the N-terminus, whereas TBAb epitopes are more diverse (11), a finding also observed with a panel of hamster monoclonal TSAbs and TBAbs (12). In 2007, the crystal structure of a human monoclonal TSAb complexed with a portion of the TSHR ectodomain was reported (13). Here it was shown that this antibody strongly interacts with TSHR residue Arg38 with the N-terminal cysteine cluster (14).

11.4.3 Detection of TSH receptor autoantibodies 11.4.3.1 Early TRAb assays The TSH receptor’s structure is known to be complex (15). In fact, TSH and TRAbs bind in the same region, and this is the precondition to measure TRAbs quantitatively using a simple competition. This was the base for all TRAb assays developed so far in which patients’ TRAbs are able to inhibit TSH binding to TSH receptor preparations.In 1974, Rees Smith and Hall described for the first time the original receptor assay for TRAb using particulate thyroid tissue from patients with GD and I-125-labeled bovine TSH (16). Since IgG preparations were needed and even physiologic serum IgG led to a significant nonspecific effect, these early TRAb assays showed a low functional and diagnostic sensitivity.

11.4.3.2 Liquid-phase TRAb assays with detergent-solubilized TSH receptors Some 30 years ago, detergent-solubilized TSH receptors were recognized as having significant advantages over particulate preparations in terms of much reduced nonspecific effects. This work paved the way for the classic serial TRAb assay described by Shewring and Rees Smith in 1982, based on porcine TSH receptors (17,18). Retrospectively, these assays meant a little revolution in the in vitro diagnostics of autoimmune thyroid diseases. From today’s viewpoint, however, a low functional and diagnostic sensitivity must be concluded despite this method‘s improved specificity. This was also true for liquid-phase TRAb assays, available from the early 1990s, using the recombinant human TSH receptor (uFig. 11.1) (19). In one and the same assay generation (detergent-solubilized TSHR preparations in liquid phase, I-125-labeled bovine TSH, polyethylene glycol [PEG] and centrifugation to separate bound and free fractions), with both assay types (recombinant human or native porcine TSHR), identical results in terms of functional and diagnostic sensitivity and specificity were obtained (19). The manufacturer calibrated this serial assay to the standard of Medical Research Council (MRC B65/122; LATS).

11.4.3.3 Solid-phase TRAb assays In the late 1990s, two groups were successful in producing monoclonal antibodies (mAbs) to the TSH receptor, which were useful for the immobilization of the receptor on plastic tubes or plate wells (in case of enzyme-linked immunosorbent assays [ELISAs]) without reducing the TSH receptor‘s conformation. The group of Rees Smith from Cardiff

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Fig.11.1: Three different assay generations for the detection of TSH receptor antibodies.

(UK) immobilized the native porcine TSH receptor (20); Vassart and colleagues from Brussels have done the same for the recombinant human TSH receptor 21. This led to a new generation of TRAb assays (solid-phase assays). Based on that, a porcine TRAb assay as well as a human TRAb assay became commercially available during follow-up. Additionally, nonisotopic TRAb assays were introduced – for example, luminescencebased human (LIA) (21) and peroxidase-based porcine TRAb assays (as ELISAs) (22). However, it was claimed in several studies that the higher sensitivity of solid-phase TRAb assays was mainly due to the use of the recombinant human rather than the native porcine TSHR. This claim is based on the impressive number of studies comparing porcine TSHR in one type of assay system (nonimmobilized TSHR and PEG to separate bound and free) with recombinant human TSHR in another type of assay system (TSHR immobilized on plastic tubes, larger columes of test sera, longer incubation times, wash steps after sample incubation, and tracer incubation) (21,23,24,25,26,27). In contrast, only a very small number of studies are available comparing both forms of TSHR in the same assay generation. One study compared the recombinant human TSHR in an isotopic assay with the native porcine TSHR in an ELISA using biotinylated bovine TSH; it obtained similar results (28). Hirooka et al compared both TSHRs within solid-phase TRAb assays and found similar sensitivity in untreated (97.7%; n = 43 of 44) and a comparable number of TRAb-positive subjects in treated GD patients (29). A third study from Okamoto et al stated that the measurement of TRAbs by solid-phase TRAb assays (porcine and human TSHR) was useful for predicting remission in their study population (30). Such similarity would be expected as the sequences of human and porcine TSH-R are essentially identical in the regions thought to be important for TRAb and TSH binding (31). Furthermore, nonisotopic procedures paved the way for automatable kits. What is true for liquid-phase assays is also true for solid-phase assays (immobilized TSHRs, larger sample volume, longer incubation times, more frequent wash steps): porcine and human TRAb assays achieved identical functional and diagnostic sensitivities and specificities (18,32,33,34). Moreover, liquid-phase as well as solid-phase TRAb assays used bovine

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TSH as tracer (uFig. 11.1). Solid-phase coated-tube TRAb assays were clearly more sensitive than the original liquid-phase (PEG-based) system in terms of functional and diagnostic sensitivity, and a reduction of specificity could be avoided (33,34,35). Both manufacturers calibrated their assays to the International Reference Preparation (IRP) of the National Institute for Biological Standards and Control (NIBSC 90/672). Consequently, “U/L” changed to “IU/L” (21,36). Actually, this IRP is coming to an end, and NIBSC pooled a new reference serum, which is actually under international multicentre evaluation.

11.4.3.4 TRAb assays based on human thyroid-stimulating monoclonal antibody M22 In 2003, Sanders et al terminated the development of a thyroid-stimulating monoclonal antibody of human origin (TSMAb) named M22 (37,38,39). M22 was produced from the lymphocytes of a patient with GD and was found to be most suitable for this new assay. Here the TSMAb serves as tracer, rather than the bovine TSH used in all liquid- and solid-phase TRAb assays. In detail, patient serum TRAb inhibits the binding of M22 to TSH receptor−coated plate wells. The TSH receptor is still of porcine origin. The assay is available as an ELISA; however, there are two different manual versions: the first is a “short version” with directly labeled M22 and subsequently a lower number of wash steps. The “full version” is characterized by indirectly labeled M22 (including biotin) and therefore includes more wash steps (40). In the latter assay, patient serum samples are incubated in (porcine) TSH receptor−coated ELISA plate wells and, after a wash step, M22-biotin is added to react with TSH receptors not occupied by testserum TRAbs. Biotinylated M22 binding to the receptor-coated wells is quantitated using streptavidin-peroxidase, and TRAbs are detected by their ability to inhibit M22biotin binding (uFig. 11.1) (41). Functional assay sensitivity (fas) (42) of the original M22-based TRAb assay was found to be 0.3 IU/L, strictly calibrated to NIBSC 90/672 (40,43). In contrast, the fas of the short version is lower owing to its assay principle and is only about 1.8 IU/L (NIBSC 90/672) (44). As a result, fas improved over three generations of TRAb assays and was about 8 U/L in liquid-phase TRAb assays, about 1.0 IU/L in solid-phase assays and, as mentioned in section 11.4.3.3, about 0.3 IU/L in M22-based TRAb assays. At present no isotopic variant of this M22-based TRAb assay is on the market, and it is doubtful that it ever will be. Recently a fully automated TRAb measurement became available for the first time. This assay is strictly calibrated to NIBSC 90/672, and a fas about 0.73 IU/L is achievable (45). The first clinical data demonstrated that the automated TRAb measurement is suitable for daily routine use (46). Based on receiver operating characteristics (ROC) plot analysis with a 99% sensitivity and specificity, the cutoff for positivity was defined as 1.75 IU/L. This cutoff is now being used in clinical routine. Within a lot-to-lot comparison, the relative differences of TRAb values over 2 IU/L were between −9% and +10%. Below this value (around the cutoff for positivity), relative differences of TRAb values reached maximal valuesof −20 up to +15% (47). The relatively high intermethod variability (note that all M22-based TRAb assays are strictly calibrated to NIBSC 90/672) is remarkable and should be kept in mind for interpreting TRAb values in daily clinical practice (48). As mentioned previously in this

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section, a new international reference preparation is currently under evaluation (see section 11.4.3.3). This should harmonize M22-based TRAb assays.

11.4.3.5 TRAbs in autoimmune thyroid diseases 11.4.3.5.1 TRAbs in Graves’ disease TSH receptor autoantibodies are principally important in the diagnosis of autoimmune hyperthyroidism. Their specificity for GD achieved nearly 100% just in liquid-phase TRAb assays, and an improvement of their specificity was neither achievable nor necessary. The sensitivity was found to be about 80% in these assays (49). Therefore the developer of more sensitive solid-phase and M22-based TRAb assays had to be aware of preventing a decrease in specificity (33,34,36). More than 95% of newly diagnosed (untreated) Graves’ patients are actually TRAb-positive in commercially available solid-phase assays (21,50,51). This high sensitivity is independent of the TSH receptor used (human or porcine) for the detection of TRAbs (see section 11.4.3.3). In fact, TRAb-negative Graves’ patients should not exist, but the lack of sensitivity might be responsible for such false-negative results. Graves’ patients supposed to be TRAb-negative (in the literature, up to 10%; however, much lower in personal experiences) have mild hyperthyroidism (52,53), and sensitive solid-phase and M22 based assays detect low but increased TRAb titers (21). Some may argue that clinical examination together with ultrasound of the neck, thyroid scintigraphy, and measurements of TSH, (free) T3, and (free) T4 are sufficient to make the diagnosis of GD in about 80% of all cases. Although this is absolutely correct, it is less practicable from the clinical point of view. Because of the high sensitivity of modern TRAb measurement technology, the relatively low cost, and the high efficacy for arriving at the diagnosis (with all its consequences for therapeutic management compared with nonautoimmune hyperthyroidism), this is much more practicable in routine clinical use. Moreover, for the diagnosis of GD, thyroid scintigraphies are frequently dispensable as well. TRAb measurement is also useful for the differentiation of hyperthyroidism from other autoimmune and nonautoimmune thyroid diseases. Here, TRAb measurement should also be performed. Furthermore, therapeutic management (radioiodine treatment and thyroidectomy planning) is also influenced by TRAb results (54,55). Another important issue to be considered is the use of TRAb measurement for the prediction of outcome in GD (56). Here we must take into account that 1. TRAb titers usually fall after the initiation of antithyroid drug therapy. 2. TRAb titers also fall after subtotal, near total, or total thyroidectomy (possibly owing to the remove of the main antigen source). 3. TRAb titers increase following radioiodine treatment (possibly because of increased secretion of the antigen in to the bloodstream and the presence of antigen-presenting cells). An excellent prospective study by Laurberg et al (57) with a follow-up of 5 years has shown these phenomena in detail. Based on this knowledge, various studies have aimed at defining cutoffs that should help clinicians to predict the outcome of GD. It was found that there is a direct correlation between TRAb concentrations and the chance of developing a relapse of GD or failing remission. It must be pointed ou, that there are also correlations between a maximal suppressed TSH serum level and

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enlarged thyroid volumes (>40 mL), respectively, and developing relapse (55,58,59). With regard to TRAbs, high serum values determined weeks after initiating antithyroid drug therapy can give reliable results in terms of predicting a potential remission. In an earlier work, we postulated a cutoff at 10 IU/L using a solid-phase assay and found the positive predictive value to be 97% (60). Similar data obtained at later time points of the disease (61,62) showed comparable results. Nonetheless, positive predictive value is still a point at issue in the literature (23,60,63) because of its dependence on the heterogeneity of the populations studied. Whereas ongoing hyperthyroidism after antithyroid drug therapy is well correlated with elevated TRAb concentrations in populations with iodine sufficiency, the situation in iodine-deficient populations is heterogeneous (32), since the functional status of the thyroid gland is influenced by antithyroid medication as well as iodine intake. However, we should consider whether it is acceptable to allow patients to become thyrotoxic again when a reliable predictor is available and the cost of extra visits to the hospital is taken into account (32,64). Thus TRAb measurement will help to identify patients for an early definitive therapy (thyroidectomy or radioiodine treatment), resulting in decreased costs for patient surveillance during antithyroid drug therapy (54,60,64).

11.4.3.5.2 TRAbs in Graves’ ophthalmopathy TRAb measurement can also be used to verify the diagnosis of Graves’ ophthalmopathy (GO) as well as to predict the outcome of GO in a certain number of patients. Usually, GO can be diagnosed easily based on typical symptoms (e.g., upper lid retraction) and associated hyperthyroidism. A special situation, however, exists in patients with GO-like symptoms without thyroid dysfunction (2%–5% of all GO patients). These patients commonly present with atypical and especially unilateral manifestations without marked inflammatory symptoms. Therefore TRAb measurement with sensitive assay technology is especially important for the differential diagnosis of unilateral exophthalmia. Based on a solid-phase (human) TRAb assay, positive TRAb levels can be found in 69% of the patients. In terms of outcome prediction of GO, TRAb measurement can also be applied. A retrospective analysis of a GD data base revealed the following results (65): Patients were classified according to GO activity and severity and grouped in terms of mild versus severe GO. GO was defined as mild when the clinical activity score (CAS) was less than 4 and the NOSCPECS score was greater than 5, whereas severe disease was defined by a CAS greater than or or equal to 4 and/or a NOSCPECS score greater than or or equal to 5. Within 4 months after the initial presentation of GO, TRAb levels below 5.7 IU/L were associated with a mild course of GO. At disease onset, higher levels did not seem to yield relevant information. Five to 8 months after initial symptoms appeared, TRAb values above 8.8 IU/L were associated with a 18.4 times elevated risk of severe disease, whereas patients with TRAb values below 2.6 IU/L had a 6.8 times higher chance of developing only mild symptoms. These data clearly demonstrate that there is a direct correlation between TRAb values and the outcome of GO at least in patients with high TRAb values.

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11.4.3.5.3 TRAbs in chronic autoimmune thyroiditis Besides measuring TSH, antibodies to thyroperoxidase (TPOAbs) and thyroglobulin (TgAbs) are the most important laboratory features in patients with chronic autoimmune thyroiditis. As far as is known, there is no direct correlation between the levels of TPOAbs (or TgAbs) and the risk for developing overt hypothyroidism. On the other hand, a population survey demonstrated that persons with overt hypothyroidism have an increased prevalence of TPOAbs (OR = 39.7) (66). With regard to TRAbs, these antibodies can also be detected in autoimmune thyroiditis. After the introduction of more sensitive solid-phase assays, it was often possible to detect TRAbs in up to 5% to 10% of patients with HT (21,34). This is not surprising in view of the autoimmune similarity between GD and HT, but it is remarkable in terms of differentiating both diagnoses. Typically TRAb concentrations in HT are at lower levels (1.5−3 IU/L) and manual liquid-phase TRAb assays are not sensitive enough to detect them (38). The differentiation of stimulating and blocking TRAbs allowed us to look into these phenomena in greater detail. Blocking TSH receptor antibodies (TSBAbs) are detectable in a a subgroup (10%−20%) of HT patients (67,68). Since, in 40% of the cases, these TSBAbs disappear and the patients achieve euthyroidism, TSBAbs seem to play a direct role in causing thyroid failure (67). It is therefore still a matter of debate whether (weakly) TRAb-positive HT patients are properly classified as having autoimmune thyroiditis with the presence of TRAbs or whether these patients should be classified as having GD but showing signs and symptoms of HT. Problematically, the cost of developing a TSBAb assay for routine clinical use seemed to be too high with regard to the relatively small group of patients who might profit from such an assay. Therefore TSBAb assays have thus far not been developed. A combined measurement of TRAbs, TPOAbs, and TgAbs is recommended in patients with chronic autoimmune thyroiditis and hypothyroidism in order to differentiate the cause of their condition. Chronic autoimmune thyroiditis is the main noniatrogenic cause of primary hypothyroidism among individuals who receive sufficient iodine in their diets. In regions where iodine deficiency is common, dietary goitrogens may be relevant, as are organification and symporter defects; inactivating TSH receptor mutations can also rarely occur (69).

11.4.3.5.4 Postpartum thyroiditis TRAb measurement is important to differentiate between GD and postpartum thyroiditis in female patients who develop hyperthyroidism postpartum. From the clinical point of view (including the use of ultrasound), it is difficult to differentiate between an initial state of postpartum thyroiditis and GD. Here, TRAb assays are easy to perform and can help to find the correct diagnosis. This is also important because thyroid scintigraphy is inadivsable for women who may be breastfeeding (14).

11.4.3.5.5 Silent thyroiditis Silent thyroiditis rarely occurs in European countries and is understood as temporary condition of HT. Consequently the diagnostic management is similar (see section 11.4.3.5.3).

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11.4.4 Thyroid peroxidase autoantibodies Thyroid peroxidase (TPO) is the major enzyme involved in thyroid hormone synthesis. TPO is also the key autoantigen in human Hashimoto’s disease, whereas thyroglobulin (Tg) predominates in animal models of thyroiditis.

11.4.4.1 Characteristics of TPOAbs 11.4.4.1.1 Classes, subclasses, and affinities of TPOAbs TPOAbs in patients’ sera are predominantly IgG with TPO-specific IgA and IgE present at low levels in some individuals. The thyroid autoantibodies are polyclonal, as reflected by the presence of several IgG subclasses and kappa and lambda light (L) chains. The concentrations of serum TPOAbs are high, in some patients attaining mg/mL (70). A diverse repertoire of human monoclonal TPOAbs has been isolated; the majority from immunoglobulin gene combinatorial libraries generated by using DNA reversetranscribed from plasma cells infiltrating Graves’or Hashimoto’s thyroid glands or draining lymph nodes. The high frequency of TPOAbs in these libraries is consistent with other evidence for the role of the thyroid as a source of TPOAbs (71).

11.4.4.1.2 Structure of TPO The structure of proteins and their posttranslational modifications may have major impacts on their recognition by the immune system. TPO is a membrane-associated protein (~107 kDa) located at the apical surface of the thyrocyte. The molecule contains an heme prosthetic group and is a glycosylated homodimer. Despite much effort, TPO crystals suitable for x-ray diffraction have not been obtained. However, insight into the structure of TPO comes from related proteins. Human TPO is 42% homologous with granulocyte myeloperoxidase (MPO) (72). MPO has been crystallized and its three-dimensional structure determined (73). The prosequence in MPO (amino acids 1–121) is absent in the expressed protein. It has also been observed that TPO N-terminal residues 1 to 109 are removed from the mature molecule. Unlike TPO, MPO is a soluble protein and homology between the two proteins ends at TPO residue 738. Within the juxtamembrane region, residues 741 to 795 resemble a complement control protein (CCP) repeat and residues 794 to 839 are homologous to the epidermal growth factor (EGF) low-density lipoprotein receptor. The CCP-like and EGF-like segments link the MPO-like domain of TPO to the plasma membrane and its short intracellular region.

11.4.4.1.3 Autoantibody recognition of TPO Serum TPOAbs interact with a restricted region on TPO, unlike a panel of mouse monoclonals that recognizes diverse epitopes (74). Monoclonal human TPOAbs (Fab) define an immunodominant region (IDR) that is recognized by sera from all individuals and comprises more than 80% of TPOAbs in the serum of an individual patient (75). Moreover, the recombinant TPO Fab binds to overlapping epitopes in two domains, A and B. Human monoclonal TPOAbs, like serum TPOAbs, preferentially bind to native rather than denatured TPO. On the other hand, some recombinant TPO polypeptide

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fragments are recognized by patients’ sera. Several linear epitopes have been described: the mAb 47 = C21 and C2 epitopes corresponding to amino acid residues 713 to 721 = 710–722 and 590 to 622, respectively (76). Human monoclonal TPOAbs representing the major component of the autoantibody repertoire were isolated using native TPO protein to screen combinatorial immunoglobulin gene libraries. To complete the molecular cloning of the repertoire of human TPOAbs, less common antibodies were obtained by screening these libraries: (a) on denatured TPO; (b) on TPO with the IDR “masked” with four recombinant human monoclonal TPOAbs (Fab); and (c) with a recombinant CCP = EGF domain polypeptide. These approaches yielded a small number of novel non-IDR TPOAbs. However, these studies emphasized the bias of the TPOAb repertoire toward recognition of conformational epitopes in an IDR.

11.4.4.1.4 Measurement of TPOAbs TPOAbs are usually measured by RIA, EIA, or LIA assays. In contrast to assays using microsomes, these modern assays use recombinant TPO for antibody detection. The sensitivity of both assay designs is equal. In contrast, however, the specificity of TPObased assays is higher. These assays are calibrated against the NIBC 66/387 standard. Reference levels are not internationally standardized.

11.4.4.1.5 TPOAbs as markers of disease One major question is whether TPOAbs may mediate thyroid dysfunction. Because TPO is the key enzyme in thyroid hormone synthesis, it is logical to consider that TPOAbs contribute to hypothyroidism by inhibiting TPO enzymatic activity. Studies using Hashimoto sera containing polyclonal TPOAbs supported this possibility. However, monoclonal human TPOAbs had no effect on TPO enzymatic activity (77). There is some evidence that TPOAbs mediate damage of thyroid follicular cells. Unlike autoantibodies to Tg (TgAbs), TPOAbs can activate the complement cascade; moreover, TPOAbs damage thyroid cells in vitro by an antibody-dependent cell cytotoxic mechanism. However, the relevance of TPOAb-mediated thyroid damage in vivo is lessened by other persuasive data for T cell–mediated thyrocyte damage in a mouse model. Thyroiditis and hypothyroidism develop spontaneously in TAZ10 transgenic mice that lack B cells (or antibody) and express the T-cell receptor of a human TPO-specific clone (78). TPOAbs mayhave a role in antigen presentation. From the clinical point of view, an even more important question is whether TPOAbs may represent markers of thyroid dysfunction. It has been demonstrated that (a) TPOAbs reflect lymphocytic infiltration of the thyroid gland, even in subclinical disease (79); (b) hypothyroidism ensues at a rate of ~2% per year in TPOAbpositive individuals (80); and (c) TPOAbs are invaluable markers of postpartum thyroiditis (81) as well as for thyroid dysfunction induced by recombinant interferon (IFN)-alpha treatment for hepatitis C (82). Another study (the NHANES survey [66]) showed that there is a strong correlation between the presence of TPOAbs and the development of hypothyroidism. The odds ratio for subclinical hypothyroidism and the presence of TPOAbs was 8.4, whereas the odds ratio for manifest hypothyroidism and the presence of TPOAbs was 39.7. In contrast, there was no significance for the presence of TgAbs and manifest hypothyroidism. Moreover, changes in TPOAb levels

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parallel those of thyrotropin receptor (TSHR) autoantibodies in Graves’ patients treated with antithyroid drugs (decrease) or radioiodine (transient increase). Overall, regardless of whether or not TPOAbs directly cause thyroid damage, they are the ideal markers for diagnosing HT and may be used to a lesser extend for outcome prediction.

11.4.5 Thyroglobulin autoantibodies 11.4.5.1 Thyroglobulin Thyroglobulin is a 670-kDa protein with two polypeptide chains of approximately 2,768 amino acids each, from which the thyroid hormones triiodothyronine (T3) and thyroxine (T4) are produced. The complete sequence of the thyroglobulin gene has been determined in humans and 40 antigenic epitopes on human thyroglobulin have been identified, with only four to six epitopes believed to be recognized by B cells and involved in the antibody response to thyroglobulin. There is evidence to suggest that iodination of thyroglobulin may alter the epitope binding patterns and that this results in multiple molecular configurations occurring naturally that are compatible with adequate hormone synthesis (i.e., that iodination of thyroglobulin results in conformational change in the molecule and change in the antigenic epitopes). Thyroglobulin has been described in the orbital tissue of patients with thyroid-associated ophthalmopathy, where it could act as a cross-reacting antigen in Graves’ ophthalmology; interestingly, it is not found in orbital tissue from normal individuals (83).

11.4.5.2 Thyroglobulin antibodies in thyroid diseases Antibodies to thyroglobulin are very common in autoimmune thyroid disease, but the major drawback to their clinical application is the lack of specificity, with 11% of the general population having detectable antibodies (84). As a result, it has been suggested that these antibodies have no role to play in the routine diagnosis of autoimmune thyroid diseases (85). It has long been recognized that endogenous antithyroglobulin antibodies have the potential to interfere with the immunometric estimation of thyroglobulin; this is the most serious technical problem in the use of thyroglobulin as a tumor marker in disseminated thyroid carcinoma, since 25% of patients have these antibodies. Although radioimmunoassay is less prone to the interfering effect of these antibodies, laboratories are keen to move away from assays that require radiolabeled constituents. Interference always causes underestimation of thyroglobulin concentration, as shown by studies on antibody-positive patients with disseminated thyroid carcinoma who have no thyroglobulin detected by immunoassay (86). The interference can occur even at low antibody concentrations, but the continued presence of antibodies in patients who have undergone a total thyroidectomy hints that the immune system is still sensing the presence of thyroglobulin, as complete ablation of thyroid tissue results in the disappearance of antibodies to all the major thyroid antigens. That is to say, as thyroid tissue is the only source of thyroglobulin; therefore any persisting antibodies must indicate the continued presence of thyroglobulin (e.g., possible metastasis). All serum samples sent for thyroglobulin estimation should be tested for the presence of antithyroglobulin antibodies, and it is recommended that laboratories should not report undetectable serum thyroglobulin values if antibodies are present and the

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method produces inappropriately low or undetectable serum thyroglobulin values in patients with disseminated thyroid carcinoma and antithyroglobulin antibodies (87). The same guidelines also recommend that precise and sensitive immunoassay methods are used in preference to recovery tests, which ‘do not reliably detect thyroglobulin antibodies and should be discouraged and eliminated, as failure to detect these antibodies could cause a delay in the treatment of metastatic disease. A number of studies note that serial estimations of antibody titers can act as surrogate tumor markers in patients with disseminated thyroid carcinoma who have antithyroglobulin antibodies. This is because the antibody titer responds to changes in thyroglobulin concentration, but the apparently paradoxical increase resulting from increased thyroglobulin antigen release due to radioactive iodine treatment must be taken into account. However, these antibody measurements must be followed up using the same manufacturer’s method, since absolute measurements differ from kit to kit despite claims that methods are standardized against the Medical Research Council 65/93 reference preparation (88). This may reflect a lack of demand or an absence of guidelines.

11.4.6 Sodium-iodide-symporter antibodies There are other less well described antigens, like the formerly cloned and characterized sodium iodide symporter, which may play a role in the pathogenesis of autoimmune thyroid disease. Sodium-iodide-symporter expression patterns are changed in autoimmune thyroid disease, including GD and HT. These may in part be caused by sodium iodide symporter regulation of the cytokine expression involved in the pathogenesis of autoimmune thyroid disease. Furthermore, there is increasing evidence that sodium iodide symporter-directed antibodies are present in serum samples from patients with autoimmune thyroid disease, and these antibodies may also affect sodium iodide symporter functional activity (89,90), although this is not a view shared by others, including our own group (91).

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45. Hermsen D, Broecker-Preuss M, Casati M et al.: Technical evaluation of the first fully automated assay for the detection of TSH receptor autoantibodies. Clin Chim 46. Schott M, Hermsen D, Broecker-Preuss M et al.: Clinical value of the first automated TSH receptor autoantibody assay for the diagnosis of Graves’ disease (GD): an international multicentre trial. Clin Endocrinol (Oxf) 2009;71(4):566–573. 47. Hermsen D, Eckstein A, Schinner S et al.: Reproducibility of Elecsys anti-TSHR test results in a lot-to-lot comparison. Horm Metab Res 2010;42(4):295–297. 48. Massart C, Sapin R, Gibassier J, Agin A, d’Herbomez M: Intermethod variability in TSHreceptor antibody measurement: implication for the diagnosis of Graves disease and for the follow-up of Graves ophthalmopathy. Clin Chem 2009;55(1):183–186. 49. Feldt-Rasmussen U: Analytical and clinical performance goals for testing autoantibodies to thyroperoxidase, thyroglobulin, and thyrotropin receptor. Clin Chem 1996;42(1):160– 163. 50. Takasu N, Oshiro C, Akamine H et al.: Thyroid-stimulating antibody and TSH-binding inhibitor immunoglobulin in 277 Graves’ patients and in 686 normal subjects. J Endocrinol Invest 1997;20(8):452–461. 51. Zouvanis M, Panz VR, Kalk WJ, Joffe BI: Thyrotropin receptor antibodies in black South African patients with Graves’ disease and their response to medical therapy. J Endocrinol Invest 1998;21(11):771–774. 52. Ilicki A, Gamstedt A, Karlsson FA: Hyperthyroid Graves’ disease without detectable thyrotropin receptor antibodies. J Clin Endocrinol Metab 1992;74(5):1090–1094. 53. Kawai K, Tamai H, Matsubayashi S et al.: A study of untreated Graves’ patients with undetectable TSH binding inhibitor immunoglobulins and the effect of anti-thyroid drugs. Clin Endocrinol (Oxf) 1995;43(5):551–556. 54. Dietlein M, Dressler J, Grunwald F et al.: [Guideline for in vivo- and in vitro procedures for thyroid diseases (version 2)]. Nuklearmedizin 2003;42(3):109–115. 55. Dietlein M, Dressler J, Grunwald F et al.: [Guideline for radioiodine therapy for benign thyroid diseases (version 4)]. Nuklearmedizin 2007;46(5):220–223. 56. Eckstein A, Mann K, Kahaly GJ et al.: [Role of TSH receptor autoantibodies for the diagnosis of Graves’ disease and for the prediction of the course of hyperthyroidism and ophthalmopathy. Recommendations of the Thyroid Section of the German Society of Endocrinology]. Med Klin (Munich) 2009;104(5):343–348. 57. Laurberg P, Wallin G, Tallstedt L, Abraham-Nordling M, Lundell G, Torring O: TSHreceptor autoimmunity in Graves’ disease after therapy with anti-thyroid drugs, surgery, or radioiodine: a 5-year prospective randomized study. Eur J Endocrinol 2008; 158(1):69–75. 58. Hoermann R, Quadbeck B, Roggenbuck U et al.: Relapse of Graves’ disease after successful outcome of antithyroid drug therapy: results of a prospective randomized study on the use of levothyroxine. Thyroid 2002;12(12):1119–1128. 59. Quadbeck B, Hoermann R, Roggenbuck U, Hahn S, Mann K, Janssen OE: Sensitive thyrotropin and thyrotropin-receptor antibody determinations one month after discontinuation of antithyroid drug treatment as predictors of relapse in Graves’ disease. Thyroid 2005;15(9):1047–1054. 60. Schott M, Morgenthaler NG, Fritzen R et al.: Levels of autoantibodies against human TSH receptor predict relapse of hyperthyroidism in Graves’ disease. Horm Metab Res 2004;36 (2):92–96. 61. Quadbeck B, Hoermann R, Hahn S, Roggenbuck U, Mann K, Janssen OE: Binding, stimulating and blocking TSH receptor antibodies to the thyrotropin receptor as predictors of relapse of Graves’ disease after withdrawal of antithyroid treatment. Horm Metab Res 2005;37(12):745–750.

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62. Carella C, Mazziotti G, Sorvillo F et al.: Serum thyrotropin receptor antibodies concentrations in patients with Graves’ disease before, at the end of methimazole treatment, and after drug withdrawal: evidence that the activity of thyrotropin receptor antibody and/or thyroid response modify during the observation period. 63. Zimmermann-Belsing T, Nygaard B, Rasmussen AK, Feldt-Rasmussen U: Use of the 2nd generation TRAK human assay did not improve prediction of relapse after antithyroid medical therapy of Graves’ disease. Eur J Endocrinol 2002;146(2):173–177. 64. Davies TF, Roti E, Braverman LE, DeGroot LJ: Thyroid controversy – stimulating antibodies. J Clin Endocrinol Metab 1998;83(11):3777–3785. 65. Eckstein AK, Plicht M, Lax H et al.: Thyrotropin receptor autoantibodies are independent risk factors for Graves’ ophthalmopathy and help to predict severity and outcome of the disease. J Clin Endocrinol Metab 2006;91(9):3464–3470. 66. Hollowell JG, Staehling NW, Flanders WD et al.: Serum TSH, T(4), and thyroid antibodies in the United States population (1988 to 1994): National Health and Nutrition Examination Survey (NHANES III). J Clin Endocrinol Metab 2002;87(2):489–499. 67. Takasu N, Yamada T, Takasu M et al.: Disappearance of thyrotropin-blocking antibodies and spontaneous recovery from hypothyroidism in autoimmune thyroiditis. N Engl J Med 1992;326(8):513–518. 68. Tamaki H, Amino N, Kimura M, Hidaka Y, Takeoka K, Miyai K: Low prevalence of thyrotropin receptor antibody in primary hypothyroidism in Japan. J Clin Endocrinol Metab 1990;71(5):1382–1386. 69. Dayan CM: Interpretation of thyroid function tests. Lancet 2001;357(9256):619–624. 70. McLachlan SM, Rapoport B.: The molecular biology of thyroid peroxidase: cloning, expression and role as autoantigen in autoimmune thyroid disease. Endocr Rev 1992;13(2):192–206. 71. McLachlan SM, McGregor A, Smith BR, Hall R: Thyroid-autoantibody synthesis by Hashimoto thyroid lymphocytes. Lancet 1979;1(8108):162–163. 72. Kimura S, Hong YS, Kotani T, Ohtaki S, Kikkawa F: Structure of the human thyroid peroxidase gene: comparison and relationship to the human myeloperoxidase gene. Biochemistry 1989;28(10):4481–4489. 73. Zeng J, Fenna RE: X-ray crystal structure of canine myeloperoxidase at 3 A resolution. 74. Ruf J, Toubert ME, Czarnocka B, Durand-Gorde JM, Ferrand M, Carayon P: Relationship between immunological structure and biochemical properties of human thyroid peroxidase. Endocrinology 1989;125(3):1211–1218. 75. Chazenbalk GD, Costante G, Portolano S, McLachlan SM, Rapoport B: The immunodominant region on human thyroid peroxidase recognized by autoantibodies does not contain the monoclonal antibody 47/c21 linear epitope. J Clin Endocrinol Metab 1993;77 (6):1715–1718. 76. Libert F, Ludgate M, Dinsart C, Vassart G: Thyroperoxidase, but not the thyrotropin receptor, contains sequential epitopes recognized by autoantibodies in recombinant peptides expressed in the pUEX vector. J Clin Endocrinol Metab 1991;73(4):857–860. 77. Nishikawa T, Jaume JC, McLachlan SM, Rapoport B: Human monoclonal autoantibodies against the immunodominant region on thyroid peroxidase: lack of cross-reactivity with related peroxidases or thyroglobulin and inability to inhibit thyroid peroxidase enzymatic activity. J Clin Endocrinol Metab 78. Quaratino S, Badami E, Pang YY et al.: Degenerate self-reactive human T-cell receptor causes spontaneous autoimmune disease in mice. Nat Med 2004;10(9):920–926. 79. Yoshida H, Amino N, Yagawa K et al.: Association of serum antithyroid antibodies with lymphocytic infiltration of the thyroid gland: studies of seventy autopsied cases. J Clin Endocrinol Metab 1978;46(6):859–862.

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12 Autoimmune primary adrenal insufficiency Matthias Schott and Jochen Seissler

12.1 General aspects and clinical features Primary adrenal insufficiency (Addison’s disease) is a rare disorder with a reported prevalence of approximately 100 per million inhabitants and a incidence of 0.4 to 0.6 per 100,000 in Europe (1). The female-to-male ratio is 1.5 to 2.0. Age at diagnosis peaks in the fourth decade of life. The disease is characterised by complete destruction or impaired function of adrenocortical cells. Autoimmune Addison’s disease (AAD) can occur as isolated disease (40%) or as part of an autoimmune polyendocrine syndrome (APS) type 1 or type 2, which may comprise other endocrine autoimmune diseases (e.g., thyroid disease, type 1 diabetes, hypoparathyroidism, celiac disease) and diseases of nonendocrine organs (hepatitis, malabsorption, alopecia, vitiligo). Addison’s disease (AD) rarely presents before the age of 10 years and, when diagnosed in childhood, is mostly part of APS I or adrenoleukodystrophy (2). Autoimmunity accounts for the majority of cases (80%–90%) in industrialized countries. Other causes are infection (10%–15%), infiltrating tumors, or rare gene defects (see the following list). Primary AD must be differentiated from secondary adrenal insufficiency induced by the administration of glucocorticoids as anti-inflammatory or immunosuppressive drugs. Etiologies of primary adrenal insufficiency: • Autoimmune adrenalitis • Infection (tuberculosis, viral infection [HIV, CMV]; fungal infection, coccidiomycosis, histoplasmosis) • Adrenal tumors (metastases of bronchial carcinoma, mammary carcinoma, lymphoma) • Adrenal hemorrhage (trauma, Waterhouse-Friderichsen syndrome) • Adrenal gland thrombosis • Drugs affecting steroid synthesis (mitotane, etomidate, ketoconazole, suramin) • Genetic syndromes (adrenoleukodystrophy, congenital adrenal hypoplasia, enzyme defects of steroid biosynthesis)

12.2 Etiology AAD is caused by an autoimmune response specifically directed against all three layers of the adrenal cortex and subsequent progressive destruction of endocrine cells. Predisposition to AAD is mediated by as yet unknown environmental factors and gene polymorphisms. The strongest risk factors are human leukocyte antigen (HLA) DR3-DQ2 (DRB1*03-DQA1*05-DQB1*02) and DR4-DQ8 (DRB1*04-DQA1*0301-DQB1*0302) as well as polymorphisms in the CTLA-4 gene and genes involved in vitamin D metabolism (3,4,5). Autoreactive T cells and antigen-presenting cells play a major role in the

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pathogenesis of adrenalitis and adrenal gland destruction (6,7). Prospective studies have demonstrated that circulating autoantibodies can appear years before the manifestations of AD. This suggests that adrenalitis is a chronic disease with slowly progressive destruction of the adrenal cortex.

12.3 Clinical features The major clinical symptoms of AD are unexplained loss of body weight, fatigue, hyperpigmentation of the skin, nausea, vomiting, abdominal pain, proneness to hypoglycemia, and low blood pressure (8). With an acute stress reaction there is an increased risk of life-threatening dehydration, hypotension, and shock (addisonian crisis). Owing to glucocorticoid and mineralocorticoid deficiency, there is hyponatremia, hyperkalemia, hypercalcemia, acidosis, anemia, and lymphocytosis. Adrenocortical insufficiency can be diagnosed by increased basal adrenocorticotropic hormone (ACTH), low glucocorticoid levels, and inadequate response after stimulation with ACTH (cortisol increase 90%) to differentiate CD from UC than using either ASCAs or atypical pANCAs alone (uTab. 13.3) (8). The addition of ALCAs, ACCAs, or AMCAs resulted in only minor improvements in differentiating CD and UC as compared with the ASCA/pANCA combination (27). In a prospective multicenter study, ASCA+/pANCA− predicted CD in 80% and ASCA−/pANCA+ UC in 64% of

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Tab. 13.3: Predictive power of atypical pANCA/ASCA combination to differentiate UC from CD patients Serologic marker

Sensitivity

Specificity

Positive predictive value

ASCA+ (CD)

27%

95%

73%

+

Atypical pANCA (UC)

31%

86%

82%

ASCA+/atypical pANCA− (CD)

24%

98%

84%

Atypical pANCA+/ASCA− (UC)

28%

89%

84%

patients with indeterminate colitis. After a 1-year follow-up, a definite diagnosis was reached in 32% (28). The diagnostic value of serologic markers can vary among different ethnic and geographic populations. Both ASCAs and atypical pANCAs were found to be less sensitive in Chinese and Japanese IBD patients. On the other hand, the prevalence of atypical pANCAs is significantly higher in Mexican UC patients compared with Caucasians (29,30).

13.4 Serologic markers and clinical phenotypes There is no association between atypical pANCAs and clinical characteristics in IBD (16). In contrast, ASCA seropositivity has been independently associated with a higher risk of stricturing (72% vs. 36%) as well as penetrating CD disease (51% vs. 27%) at the time of diagnosis and during follow-up as compared with patients who do not have stenosing or penetrating disease (31,32). The presence of anti-OmpC, anti-I2, and anti-CBir1 in CD patients is associated with an increased prevalence of stenosing and penetrating forms and shows a more aggressive phenotype of CD disease with a higher risk for surgical intervention (33,34,35). In a prospective study in pediatric CD patients, anti-OmpC, anti-I2, antiCBir1, and ASCA=seronegative patients remained complication-free during follow-up (33). In CD patients positive for two of ALCAs, ACCAs, and AMCAs, small intestinal disease can be detected in 93% as compared with seronegative patients (60%) (27). In UC, no clearly differences of the clinical phenotype between seropositive or seronegative patients were found for any antibody (36).

13.5 Serologic markers in disease monitoring Serial measurement of ASCAs or atypical pANCAs in IBD is not useful for disease monitoring. There is no association between the presence of atypical pANCAs and UC activity. In addition, UC patients remain atypical pANCA-positive after colectomy (37,38). The presence of ASCAs is independent of CD activity and disease progression. ASCA titers remain stable after therapy (39).

13.6 Therapeutic value of serologic markers The presence of atypical pANCAs is associated with resistance to the treatment of leftsided UC (40). Furthermore, positivity for atypical pANCAs in UC patients was

13.7 Subclinical value of serological markers

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associated with a significantly higher risk of receiving azathioprine treatment during a 10-year follow-up (8). There is no association between ASCAs and atypical pANCA positivity and response to anti−tumor necrosis factor alpha (TNF-α) or mesalamine therapy (41,42). Positivity of ASCAs, atypical pANCAs, anti-I2, and anti-OmpC remained unchanged following treatment with mesalamine, steroids, or anti−TNF-α therapy (43).

13.7 Subclinical value of serological markers An increased seroprevalence of ASCAs (20%–25%) and anti-OmpC in unaffected firstdegree relatives of CD patients has been described (44,45,46). The prevalence of ASCAs within pure CD families in terms of the number of family members affected is as follows: two members affected, 54%; three members affected, 75% (39). ASCAs and atypical pANCAs precede the clinical diagnosis of CD and UC by about 38 months (47). However, large prospective studies remain to be performed to confirm these data.

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42. Papp M, Altorjay I, Dotan N, Palatka K, Foldi I, Tumpek J, Sipka S, Udvardy M, Dinya T, Lakatos L, Kovacs A, Molnar T, Tulassay Z, Miheller P, Norman GL, Szamosi T, Papp J; Hungarian IBD Study Group, Lakatos PL. New serological markers for inflammatory bowel disease are associated with earlier age at onset, complicated disease behavior, risk for surgery, and NOD2/CARD15 genotype in a Hungarian IBD cohort. Am J Gastroenterol 2008;103:665–681. 43. Teml A, Kratzer V, Schneider B, Lochs H, Norman GL, Gangl A, Vogelsang H, Reinisch W. Anti-Saccharomyces cerevisiae antibodies: a stable marker for Crohn’s disease during steroid and 5-aminosalicylic acid treatment. Am J Gastroenterol 2003;98:2226–2231. 44. Seibold F, Stich O, Hufnagl R, Kamil S, Scheurlen M: Anti-Saccharomyces cerevisiae antibodies in inflammatory bowel disease: a family study. Scand J 45. Joossens M, Van Steen K, Branche J, Sendid B, Rutgeerts P, Vasseur F, et al.: Familial aggregation and antimicrobial response dose-dependently affect the risk for Crohn’s disease. Inflamm Bowel Dis 2010;16:58–67. 46. Sutton CL, Yang H, Li Z, Rotter JI, Targan SR, Braun J: Familial expression of antiSaccharomyces cerevisiae mannan antibodies in affected and unaffected relatives of patients with Crohn’s disease. Gut 2000;46:58–63. 47. Israeli E, Grotto I, Gilburd B, Balicer RD, Goldin E, Wiik A, et al.: Anti-Saccharomyces cerevisiae and antineutrophil cytoplasmic antibodies as predictors of inflammatory bowel disease. Gut 2005;54:1232–1236.

14 Celiac disease Thomas Griga

14.1 General aspects and clinical features Celiac disease is an autoimmune disorder of the upper small intestine in genetically predisposed patients induced by the ingestion of gluten. Untreated classic celiac disease leads to the characteristic symptoms of malabsorption, with chronic diarrhea, weight loss, and anemia (1). There are also silent or atypical forms presenting with nonspecific abdominal pain, elevated liver enzyme levels, iron-deficiency anemia, and osteoporosis (2,3). Celiac disease is associated with other autoimmune or genetically determined disorders such as dermatitis herpetiformis (10%), type 1 diabetes mellitus (6%), primary biliary cirrhosis (3%), autoimmune thyroiditis (3%), peripheral neuropathy (9%), Down‘s syndrome (5%–12%), and Turner’s syndrome (3%)(4,5). The prevalence is estimated at 0.5% to 1.0% (6,7), the median delay in diagnosis ranges from 4.9 to 11 years (8,9). The ratio of known to undiagnosed cases of celiac disease is 1:7; two to three times as many women as men have the disease (10). Patients with celiac disease have an increased risk of enteropathy-associated T-cell lymphoma and adenocarcinoma of the small intestine (7,11,12). In patients with celiac disease, immune reactions to gliadin fractions induce a chronic inflammatory disorder, primarily in the upper small intestine, histologically characterized by intraepithelial lymphocytosis and villous atrophy (13,14). The adaptive response is mediated by gliadin-reactive CD4+ T cells in the lamina propria, subsequently producing proinflammatory cytokines (15). There is a strong association to the HLA-DQ2 and HLA-DQ8 allele. Furthermore, numerous T cell−derived cytokines are involved in the development of celiac disease (16,17).

14.2 Diagnostic workflow As shown in uFig. 14.1, patients with chronic diarrhea, weight loss, or anemia are suspected to have a high risk for celiac disease and should have a duodenal biopsy taken according to the guidelines of the British Society of Gastroenterology (18). The other patients presenting with atypical symptoms of celiac disease − such as abdominal pain, dyspepsia, and elevated liver enzymes − are classified as at low risk. These patients should first be screened for celiac disease with serologic testing.

14.2.1 Duodenal biopsy Duodenal biopsy demonstrating raised intraepithelial lymphocyte count, crypt hyperplasia, and villous atrophy according to the modified Marsh criteria is the gold standard to confirm the diagnosis of celiac disease, especially in patients with classic symptoms of disease (uFig. 14.2a and 14.2b, uTab. 14.1) (13,14,19).

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Fig. 14.1: Diagnostic flowchart for celiac disease.

Fig. 14.2: a–b Small intestine biopsy with characteristic findings of celiac disease (intraepithelial lymphocytosis, crypt hyperplasia, and villous atrophy) (a), compared to normal mucosa (b) (courtesy of Prof. Dr. Tannapfel, Institute of Pathology, Ruhr-University Bochum).

14.2 Diagnostic workflow

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Tab. 14.1: Intestinal lesions in celiac disease Type

Histologic findings

Marsh 0 preinfiltrative stage

Normal

Marsh 1 infiltrative lesion

Increased intraepithelial lymphocytes

Marsh 2 hyperplastic lesion

Plus hyperplastic crypts

Marsh 3 destructive lesion

Plus partial villous atrophy (3a), subtotal villous atrophy (3b), total villous atrophy (3c)

Marsh 4 hypoplastic lesion

Total villous atrophy with crypt hypoplasia

The histologic findings in celiac disease are characteristic but not specific (see the following list). The differential diagnosis of villous atrophy in celiac disease is as follows: • • • • • • • •

Collagenous sprue Eosinophilic enteritis Intestinal lymphoma Tuberculosis Crohn’s disease HIV enteropathy Autoimmune enteropathy Giardiasis

14.2.2 Serologic testing Patients having a low risk with nonspecific symptoms or who are unable to undergo gastroscopy with duodenal biopsy should first be tested serologically. It is important to remain on a gluten-containing diet before celiac disease−associated antibody testing begins (20). Furthermore, it must be taken in consideration that approximately 6.4% of all cases of celiac disease are antibody-negative (21).

14.2.2.1 Tissue transglutaminase antibody (tTG) IgA The enzyme tissue transglutaminase (tTG) is the autoantigen for the development of endomysial antibodies (22). Measurement of serum concentration of tTG immunoglobulin A (IgA) is recommended for initial testing because of its high sensitivity (100%) and specificity (97%) for celiac disease, relatively low cost, and ease of test performance as well as reliability (23,24). However, the sensitivity and specificity differ among laboratories (25). False-positive test results may occur in patients with chronic liver disease (26,27). If the serum concentration of tTG IgA antibodies is normal or absent and there is high suspicion of celiac disease, the total IgA level should be measured to rule out selective IgA deficiency, which is more common in patients with celiac disease (1:40) than in the general population (28,29) (1:600). In such cases, tTG IgG antibodies or duodenal biopsy should be performed (30). If the total IgA level is normal, patients should be screened for HLA DQ2 or DQ8.

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14.2.2.2 Human leukocyte antigen (HLA) testing Human leukocyte antigen (HLA)-DQ2 (encoded by HLA-DQA1*0501 or *0505 alleles and HLA-DQB1*0201 or *0205 alleles) is found in more than 90% of patients with celiac disease and in 20% to 30% of the general population. HLA-DQ8 (encoded by HLADQA1*03 and HLA-DQB1*0302 alleles) is found in 5% to 10% of individuals with celiac disease and approximately 10% of the general population (31,32). Virtually 100% of celiac disease patients are positive for one of these alleles. Therefore a negative HLA screen can exclude celiac disease in patients with suspicion of celiac disease if the serum concentration of tTG IgA antibodies is normal or absent and the total IgA level is normal (21,31,33). Unlike the outcomes of antibody testing and duodenal biopsy, the results of molecular genetic testing can be interpreted accurately independent of diet.

14.2.2.3 Endomysial antibody (EMA) IgA The sSerum concentration of endomysial antibody (EMA) IgA has the highest specificity (99%) but is more expensive, labor-intensive, and potentially more prone to falsenegative results than the serum concentration of tTG IgA (34). Because it is determined by indirect immunofluorescence, the serum concentration of EMA IgA is subjective, which affects its sensitivity (35). When performed in an experienced laboratory, this test has a higher specificity (100%) than the tTG antibody test and should be performed in patients with liver cirrhosis. Furthermore, EMA IgA should be investigated in individuals with positive tTG IgA screening confirming the diagnosis of celiac disease if duodenal biopsy could not be performed.

14.2.2.4 Antigliadin antibodies (AGAs) IgA and IgG Antigliadin antibodies (AGAs) are no longer considered sensitive or specific enough to be used for the diagnosis of celiac disease (36).

14.2.2.5 Deaminated gliadin peptide (DGP) IgG and IgA More recently, deaminated gliadin peptide (DGP) antibodies especially of the IgG class have been introduced with sensitivity and specifity comparable to tTG antibodies and EMA, but improving the performance in IgA-deficient patients and in children (37).

14.2.3 Persistent antibodies and refractory disease The persistence of tTG antibodies and EMA in patients on a gluten-free diet for 1 year or more is suggestive of poor dietary adherence (20). Approximately 5% of patients have refractory celiac disease with persistent symptoms and villous atrophy despite dietary adherence (38). In such cases intraepithelial lymphocyte phenotyping should be performed, since an aberrant population (CD3+, CD8-) is associated with a high risk of T-cell lymphoma of the small intestine (39,40,41).

References 1. Green PH, Cellier C: Celiac disease. N Engl J Med 2007;357:1731–1743. 2. Telega G, Bennet TR, Werlin S: Emerging new clinical patterns in the presentation of celiac disease. Arch Pediatr Adolesc Med 2008;162:164–168.

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3. Sanders DS, Hopper AD, Azmy IA, Rahman N, Hurlstone DP, Leeds JS, et al. Association of adult celiac disease with surgical abdominal pain: a case-control study in patients referred to secondary care. Ann Surg 2005;242:201–207. 4. Green PH: The many faces of celiac disease: clinical presentation of celiac disease in the adult population. Gastroenterology 2005;128:S74–S78. 5. Kingham J, Parker D: The association between primary biliary cirrhosis and celiac disease: a study of relative prevalences. Gut 1998;42:120–122. 6. Fasano A, Berti I, Gerarduzzi T, Not T, Colletti RB, Drago S, et al.: Prevalence of celiac disease in at-risk and not-at-risk groups in the United States. Arch Intern Med 2003; 163:286–292. 7. Green PH, Jabri B: Celiac disease. Lancet 2003;362:383–391. 8. Green PHR, Stavropoulos SN, Panagi SG, Goldstein SL, Mcmahon DJ, Absan H, et al.: Characteristics of adult celiac disease in the USA: results of a national survey. Am J Gastroenterol 2001;96:126–131. 9. Sanders DS, Hurlstone DP, Stokes RO, Rashid F, Milford-Ward A, Hadjivassiliou M, et al.: Changing face of adult celiac disease: experience of a single university hospital in South Yorkshire. Postgrad Med J 2002;78:31–33. 10. Fasano A, Catassi C: Current approaches to diagnosis and treatment of celiac disease: an evolving spectrum. Gastroenterology 2001;120:636–651. 11. Howdle PD, Jalal PK, Holmes GK, Houlston RS: Primary small-bowel malignancy in the UK and its association with celiac disease. QJM 2003;96:345–353. 12. Green PH, Fleischauer AT, Bhagat G, Goyal R, Jabri B, Neugut AI: Risk of malignancy in patients with celiac disease. Am J Med 2003;115:191–195. 13. Marsh MN: Gluten, major histocompatibility complex, and the small intestine. A molecular and immunobiologic approach to the spectrum of gluten sensitivity (‘celiac sprue’). Gastroenterology 1992;102:330–334. 14. Hill ID, Dirks MH, Liptak GS, Colletti RB, Fasano A, Guandalini S, et al. North American Society for Pediatric Gastroenterology, Hepatology and Nutrition: Guideline for the diagnosis and treatment of celiac disease in children: recommendations of the North American Society for Pediatric Gastroenterology, Hepatology and Nutrition. J Pediatr Gastroenterol Nutr 2005;40:1–19. 15. Sollid LM: Celiac disease: dissecting a complex inflammatory disorder. Nat Rev Immunol 2002;2:647–655. 16. De Re V, Simula MP, Caggiari L, Ortz N, Spina M, Da Ponte Aet al.: Proteins specifically hyperexpressed in a celiac disease patient with aberrant T cells. Clin Exp Immunol 2007; 148:402–409. 17. Kolkowski EC, Fernandez MA, Pujol-Borrell R, Jaraquemada D: Human intestinal alphabeta IEL clones in celiac disease show reduced IL-10 synthesis and enhanced IL-2 production. Cell Immunol 2006;244:1–9. 18. Holbrook I: The British Society of Gastroenterology guidelines for the investigation of chronic diarrhoea, 2nd edition. Ann Clin Biochem 2005;42:170–174. 19. National Institutes of Health Consensus Development Conference Statement on Celiac disease. Gastroenterology 2005;128:S1–S9 20. Midhagen G, Aberg AK, Olce´n P, Ja¨rnerot G, Valdimarsson T, Dahlbom I, et al.: Antibody levels in adult patients with coeliac disease during gluten-free diet: a rapid initial decrease of clinical importance. J Intern Med 2004;256:519–524. 21. Collin P, Kaukinen K, Vogelsang H, Korponay-Szabo´ I, Sommer R, Schreier E, et al.: Antiendomysial and antihuman recombinant tissue transglutaminase antibodies in the diagnosis of celiac disease: a biopsy-proven European multicentre study. Eur J Gastroenterol Hepatol. 2005;17:85–91. 22. Dieterich W, Ehnis T, Bauer M, Donner P, Volta U, Riecken EO, et al.: Identification of tissue transglutaminase as the autoantigen of celiac disease. Nat Med 1997;3:797–801.

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23. Rostom A, Murray JA, Kagnoff MF. American Gastroenterological Association (AGA) Institute: technical review on the diagnosis and management of celiac disease. Gastroenterology 2006;131:1981–2002. 24. Hopper AD, Cross SS, Hurlstone DP, McAlindon ME, Lobo AJ, Hadjivassiliou M, et al.: Pre-endoscopy serological testing for celiac disease: evaluation of a clinical decision tool. BMJ 2007;334:729. 25. Abrams JA, Brar P, Diamond B, Rotterdam H, Green PH: Utility in clinical practice of immunoglobulin A anti-tissue transglutaminase antibody for the diagnosis of celiac disease. Clin Gastroenterol Hepatol 2006;4:726–730. 26. Germenis AE, Yiannaki EE, Zachou K, Roka V, Barbanis S, Liaskos C, et al.: Prevalence and clinical significance of immunoglobulin A antibodies against tissue transglutaminase in patients with diverse chronic liver diseases. Clin Diagn Lab Immunol 2005;12:941– 948. 27. Lo Iacono O, Petta S, Venezia G, Di Marco V, Tarantino G, Barbaria F, et al.: Anti-tissue transglutaminase antibodies in patients with abnormal liver tests: is it always coeliac disease? Am J Gastroenterol 2005;100:2472–2477. 28. Alaedini A, Green PH: Narrative review: celiac disease: understanding a complex autoimmune disorder. Ann Intern Med 2005;142:289–298. 29. Wong RC, Steele RH, Reeves GE, Wilson RJ, Pink A, Adelstein S: Antibody and genetic testing in coeliac disease. Pathology 2003;35:285–304. 30. Cataldo F, Lio D, Marino V, Picarelli A, Ventura A, Corazza GR: IgG(1) antiendomysium and IgG antitissue transglutaminase (anti-tTG) antibodies in coeliac patients with selective IgA deficiency. Working Groups on Celiac Disease of SIGEP and Club del Tenue. Gut 2000;47:366–369. 31. Sollid LM, Lie BA: Celiac disease genetics: current concepts and practical applications. Clin Gastroenterol Hepatol 2005;3:843–851. 32. Liu E: Genetic testing for celiac disease. MLO Med Lab Obs 2006;38:10–13. 33. Zubillaga P, Vidales MC, Zubillaga I, Ormaechea V, Garcı´a-Urkı´a N, Vitoria JC: HLADQA1 and HLA-DQB1 genetic markers and clinical presentation in celiac disease. J Pediatr Gastroenterol Nutr 2002;34:548–554. 34. Lewis NR, Scott BB: Systematic review: the use of serology to exclude or diagnose coeliac disease (a comparison of the endomysial and tissue transglutaminase antibody tests). Aliment Pharmacol Ther 2006;24:47–54. 35. Murray J, Watson T, Beverlee C, Mitros F: Effect of a gluten-free diet on gastrointestinal symptoms in celiac disease. Am J Clin Nutr 2004;79:669–673. 36. Rostom A, Dube´ C, Cranney A, Saloojee N, Sy R, Garritty C, et al.: The diagnostic accuracy of serologic tests for celiac disease: a systematic review. Gastroenterology 2005; 128:S38–S46. 37. Mozo L, Gomez J, Escanlar E, Bousono C, Gutierrez C: Diagnostic value of antideaminated gliadin peptide IgG antibodies for celiac disease in children and IgA deficient patients. J Pediatr Gastroenterol Nutr 2012; in press. 38. Trier JS: Celiac sprue. N Engl J Med 1991;325:1709–1719. 39. Krauss N, Schuppan D: Monitoring nonresponsive patients with celiac disease. Gastrointest Endosc Clin N Am 2006;16:317–327. 40. Cellier C, Delabesse E, Helmer C, Patey N, Matuchansky C, Jabri B, et al.: Refractory sprue, coeliac disease, and enteropathy-associated T-cell lymphoma. French Coeliac Disease Study Group. Lancet 2000;356:203–208. 41. Cellier C, Patey N, Mauvieux L, Jabri B, Delabesse E, Cervoni JP, et al.: Abnormal intestinal intraepithelial lymphocytes in refractory sprue. Gastroenterology 1998;114:471–481.

15 Autoimmune polyendocrine syndromes Matthias Schott and Jochen Seissler

15.1 General aspects and clinical features Autoimmune diseases frequently affect not only a single tissue system but several organ systems simultaneously. The concomitant appearance of two or more autoimmune endocrinopathies defines the so-called autoimmune polyendocrine syndromes (APS) (synonym: polyglandular autoimmune syndrome) (1,2,3). APS is thought to be caused by the induction of autoreactive T lymphocytes against specific autoantigens, followed by tissue infiltration with CD4-positive T-helper 1 and T-helper 2 cells, CD8-positive T cells, and antigen-presenting cells (macrophages, dendritic cells). The proinflammatory milieu, cytotoxic cells, and defects in immunosuppressive regulatory T cells promote chronic progressive destruction of affected organs (4,5). Exceptions are Graves’ disease and myasthenia gravis, where the appearance of autoantibodies against the TSH receptor and the acetylcholine receptor are directly pathogenic. According to the clinical presentation and the genetic association, APS is classified into two major subtypes, types 1 and 2 (1,2,3). APS-1 and APS-2 are distinguished by age of disease manifestation, different modes of inheritance, and characteristic patterns of disease combinations.

15.2 Autoimmune polyendocrine syndrome type 1 (APS-1) Autoimmune polyendocrine syndrome type 1 (APS-1) is a rare monogenic autosomal recessive disease characterized by the concomitant appearance of at least two of three diseases: chronic mucocutaneous candidiasis (CMC), primary hypoparathyroidism (pHPT), and autoimmune Addison’s disease (AAD). According to the clinical presentation, the syndrome is also termed polyendocrinopathy-candidiasis-ectodermaldystrophy syndrome (APECED syndrome). APS-1 peaks at age 3–5 years (3,4,5), with a female preponderance (the female:male ratio is 1.5:2.4). Autoimmunity against other endocrine and nonendocrine organs is also observed, with a variable picture. The number of autoimmune diseases varies between two and eight (6,7,8,9). In almost all cases recurrent or chronic mucocutaneous candidiasis and ectodermal dystrophy (dental enamel hypoplasia, nail dystrophy, or keratopathy) are detectable. CMC is considered to be the expression of a selective immunologic defect in the uptake of Candida albicans by antigen-presenting cells, resulting in lack of initiation and maintenance of a protective T-cell response (10). The most frequent autoimmune manifestation is primary autoimmune hypoparathyroidism (AHPT) (80%–85%), which usually develops before the age of 10 years, followed by autoimmune Addison’s disease (AAD) (30%–70%). After puberty, about 60% females and 15% males develop primary gonadal deficiency. In some patients severe complications such as fulminant hepatitis, a systemic candidiasis, and carcinoma of the oral mucosa have been described. The

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autoantigens are mostly enzymes of steroid biosynthesis and liver metabolism, such as 21-hydroxylase (21OH), 17-hydroxylase (17OH), side-chain-cleavage enzyme (SCC), cytochromes P450 2A6 and 1A2 (11,12,13,14), and enzymes involved in the synthesis of neurotransmitters, such as aromatic L-amino-acid-decarboxylase (AADC), tryptophan hydroxylase (TPH), tyrosine hydroxylase (TH), and histidine hydroxylase (15,16,17,18,19). The spectrum and prevalence of autoimmune diseases are given in uTab. 15.1. In 1997 the APS-1 gene has been identified and termed autoimmune regulator (AIRE) (20,21). AIRE appears to function as a transcription factor, which regulates expression of autoantigens on specialized cells in the thymus called medullary epithelial cells; these are involved in central tolerance and negative selection. When the AIRE gene

Tab. 15.1: Characteristics and components of autoimmune polyendocrine syndrome type 1 (APS-1) and type 2 (APS-2) APS-1

APS-2

Inheritance

Autosomal recessive AIRE gene

Polygene HLA class II, CTLA-4 PTPN22, MICA

Age at presentation

0–10 years

30–50 years

Gender (female:male)

1.4 :1

3:1

Mucocutaneous candidiasis

90%–100%

Absent

Autoimmune hypoparathyroidism

76%–93%

Rare (0%–3%)

Autoimmune Addison’s disease

72%–100%

100%

Autoimmune thyroid disease

1%–11%

70%–83%

Type 1 diabetes

Rare in childhood Adults: 2%–12%

30%–60%

Hypergonadotrophic hypogonadism

17%–60%

4%–10%

Vitiligo

8%–22%

5%–11%

Alopecia

29%–37%

5%

Pernicious anemia

13%–19%

0.2%–1%

Coeliac disease

10%–12%

3%–10%

Autoimmune hepatitis

12%–20%

1%–4%

Intestinal dysfunction (diarrhea, steatorrhea, constipation)

15%–25%