Karsten Conrad

Challenges of automated screening and differentiation of non-organ specific autoantibodies on HEp-2 cells.

Analysis of autoantibodies (AAB) by indirect immunofluorescence (IIF) remains the hallmark of diagnosing autoimmune diseases despite the introduction of multiplex techniques. Non-organ specific AAB are screened in routine diagnostics by IIF on HEp-2 cells. However, IIF results vary due to objective (e.g., cell fixation) and subjective factors (e.g., expert knowledge). Therefore, inter- and intralaboratory variance is relatively high. Standardisation of AAB testing by IIF remains a critical issue in and between routine laboratories and may be improved by automated interpretation systems. An overview of existing interpretation techniques will be given taking into account own data of the first fully automated reading system AKLIDES. The novel system provides fully automated reading of IIF images and software algorithms for the mathematical description of IIF AAB patterns. It can be used for screening and preclassification of non-organ specific AAB in routine diagnostics regarding systemic autoimmune and autoimmune liver diseases. Furthermore, this system paves the way for economic data processing of cell-based IIF assays and can contribute to the reduction of interlaboratory variance of AAB testing. More sophisticated pattern recognition algorithms and novel calibration systems will improve standardised quantifications of IIF image interpretation.

Identification of GP2, the major zymogen granule membrane glycoprotein, as the autoantigen of pancreatic antibodies in Crohn’s disease

Backround and aims: The aetiopathogenesis of Crohn’s disease, an inflammatory bowel disease (IBD), is not yet fully understood. Autoimmune mechanisms are thought to play a role in the development of Crohn’s disease, but the target antigens and the underlying pathways have not been sufficiently identified. Methods: Based on data from immunoblotting and matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry, the major antigenic target of pancreatic autoantibodies (PABs), which are specific for Crohn’s disease, was identified. Specificity of autoantibody reactivity was confirmed by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IIF) using purified rat and human recombinant GP2 synthesised in transiently transfected mammalian HEK 293 cells. Real-time polymerase chain reaction (rt-PCR) and IIF were used to detect mRNA and antigen localisation in human colon biopsies. Results: The major zymogen granule membrane glycoprotein 2 (GP2) was identified as the autoantigen of PABs in Crohn’s disease. PAB-positive sera from patients with Crohn’s disease (n = 42) displayed significantly higher IgG reactivity to rat GP2 in ELISA than either PAB-negative sera (n = 31), or sera from patients with ulcerative colitis (n = 49), or sera from blood donors (n = 69) (p<0.0001, respectively). Twenty-eight (66%) and 18 (43%) of 42 PAB-positive sera demonstrated IgG and IgA reactivity to human recombinant GP2 in IIF, respectively. Patients with PAB-negative Crohn’s disease (n = 31) were not reactive. GP2 mRNA transcription was significantly higher in colon biopsies from patients with Crohn’s disease (n = 4) compared to patients with ulcerative colitis (n = 4) (p = 0.0286). Immunochemical staining confirmed GP2 expression in human colon biopsies from patients with Crohn’s disease. Conclusion: Anti-GP2 autoantibodies constitute novel Crohn’s disease-specific markers, the quantification of which could significantly improve the serological diagnosis of IBD. The expression of GP2 in human enterocytes suggests an important role for anti-GP2 response in the pathogenesis of Crohn’s disease.

Interleukin-17-producing T helper cells in autoimmunity

With all the incredible progress in scientific research over the past two decades, the trigger of the majority of autoimmune disorders remains largely elusive. Research on the biology of T helper type 17 (T(H)17) cells over the last decade not only clarified previous observations of immune regulations and disease manifestations, but also provided considerable information on the signaling pathways mediating the effects of this lineage and its seemingly dual role in fighting the invading pathogens on one hand, and in frightening the host by inducing chronic inflammation and autoimmunity on the other hand. In this context, recent reports have implicated T(H)17 cells in mediating host defense as well as a growing list of autoimmune diseases in genetically-susceptible individuals. Herein, we summarize the current knowledge on T(H)17 in autoimmunity with emphasis on its differentiation factors and some mechanisms involved in initiating pathological events of autoimmunity.

Diagnosis and classification of Crohn's disease

Crohns disease (CrD) is a chronic relapsing inflammatory bowel disease (IBD) potentially affecting any portion of the gastrointestinal tract from the mouth to the anus. CrD usually manifests between 15 and 30 years of age and presents typically with abdominal pain, fever, bloody or non-bloody diarrhoea, and weight loss. Paediatric patients may show failure to thrive, growth impairment, and delayed puberty additionally. Extraintestinal manifestations like arthritis, uveitis, and erythema nodosum are diagnosed in almost half of the patients. CrD is characterized by a discontinuous and ulcerous transmural inflammation often involving the ileocaecal region and leading to a stricturing or even fistulising phenotype in up to 50% of patients finally. Incidence and prevalence of CrD have been rising worldwide over the past decades. Although many details of the pathophysiology of CrD have been elucidated, no common aetiopathogenic model exists for all forms of CrD, presenting more an umbrella term for a phenotypically and genotypically heterogeneous clinical condition. In CrD, we see an inappropriate response of the innate and/or adaptive immune system to the intestinal microbiota in genetically predisposed individuals. The diagnosis of CrD is based mainly on patients history and clinical examination and supported by serologic, radiologic, endoscopic, and histologic findings. Antibodies to Saccharomyces cerevisiae and autoantigenic targets such as glycoprotein 2 may aid in differentiating CrD from UC. Their single use, however, is limited by low sensitivity requiring antibody profiling for an appropriate serologic diagnosis. This review focuses on diagnostic and classification criteria of CrD.

Defective removal of ribonucleotides from DNA promotes systemic autoimmunity

Genome integrity is continuously challenged by the DNA damage that arises during normal cell metabolism. Biallelic mutations in the genes encoding the genome surveillance enzyme ribonuclease H2 (RNase H2) cause Aicardi-Goutières syndrome (AGS), a pediatric disorder that shares features with the autoimmune disease systemic lupus erythematosus (SLE). Here we determined that heterozygous parents of AGS patients exhibit an intermediate autoimmune phenotype and demonstrated a genetic association between rare RNASEH2 sequence variants and SLE. Evaluation of patient cells revealed that SLE- and AGS-associated mutations impair RNase H2 function and result in accumulation of ribonucleotides in genomic DNA. The ensuing chronic low level of DNA damage triggered a DNA damage response characterized by constitutive p53 phosphorylation and senescence. Patient fibroblasts exhibited constitutive upregulation of IFN-stimulated genes and an enhanced type I IFN response to the immunostimulatory nucleic acid polyinosinic:polycytidylic acid and UV light irradiation, linking RNase H2 deficiency to potentiation of innate immune signaling. Moreover, UV-induced cyclobutane pyrimidine dimer formation was markedly enhanced in ribonucleotide-containing DNA, providing a mechanism for photosensitivity in RNase H2-associated SLE. Collectively, our findings implicate RNase H2 in the pathogenesis of SLE and suggest a role of DNA damage-associated pathways in the initiation of autoimmunity.

Diagnosis and classification of ulcerative colitis

Ulcerative colitis (UC) is a chronic relapsing inflammatory bowel disease (IBD) characterised by superficial mucosal ulceration, rectal bleeding, diarrhoea, and abdominal pain. In contrast to Crohns disease (CrD), UC is restricted to the colon and the inflammation is limited to the mucosal layer. Classic UC affects the colon in a retrograde and continuous fashion starting from the rectum and extending proximally. Dependent on the anatomic extent of involvement, UC can be classified as proctitis, left-sided colitis, or pancolitis. Inflammatory arthropathies and primary sclerosing cholangitis (PSC) are the most common and clinically most important extraintestinal manifestations of UC. The aetiopathogenesis of UC is incompletely understood, but immune-mediated mechanisms are responsible for dysregulated immune responses against intraluminal antigens in genetically predisposed individuals. The diagnosis is based on the history, as well as clinical, radiological, endoscopic and histological features. Autoantibodies, mainly antineutrophil cytoplasmic antibodies (ANCA) and anti-goblet cell antibodies (GAB) may be helpful in the early diagnosis of UC and in differentiating it from CrD.

Systemic lupus erythematosus after heavy exposure to quartz dust in uranium mines: clinical and serological characteristics

Antimalarial drugs are effective disease modifying agents in RA with a low incidence of serious toxic effects. Recently, combinations of second-line agents have been used in RA in attempts to treat patients with no response to a number of single agents, or suboptimal response to a single agent. Combinations of drugs have been selected for maximum efficacy and minimum toxicity, but clinical trials are difficult to design and interpret. In particular, ensuring adequate power to detect small differences in response poses a major problem. Antimalarials are an attractive choice for combination therapy due to their efficacy, mechanisms of action and toxicity profile. In this review, the evidence for the use of antimalarials in combination in RA is examined. No advantage has been shown in combining antimalarials with gold, penicillamine or sulphasalazine compared with monotherapeutic regimens. There is some evidence to suggest a beneficial combination of antimalarials with methotrexate, but this is as yet inconclusive. Open non-randomised uncontrolled studies have shown that antimalarials combined with cytotoxic agents are effective but highly toxic. The authors conclude that there is little good evidence to support the introduction of combination second-line drug therapy for RA into widespread therapeutic use.Epidemiological, clinical and serological data of uranium miners with symptoms of connective tissue diseases (CTD) were collected during the control examinations for occupational lung diseases since 1975. Twenty eight definite (four or more ARA criteria) and 15 probable (2-3 ARA criteria) SLE were diagnosed. The estimated prevalence among heavily silica exposed uranium miners was up to 93 in 100,000. The only significant differences to nonexposed SLE patients were decreased frequency of arthritis and photosensitivity and the absence of anti-Sm and anti-U1-RNP antibodies. ANA were found in all definite SLE patients examined with the following specificities: anti-dsDNA (in 44.4%), & anti-Ro/SSA (in 55.6%, four cases together with anti-dsDNA) and anti-La/SSB (in 22.2%). The autoantibody profiles of patients with probable SLE were similar, but with a lower frequency of ANA, anti-dsDNA and anti-Ro/SSA. Middle to high-titred autoantibodies to dsDNA, Ro/SSA and La/SSB were detected in 3.2% uranium miners with no (N = 1229) and in 20.6% with some symptoms (one ARA criterion and/or two or more of other CTD typical symptoms, N = 68) of CTD development. We conclude, that the strong exposure to dust with a high content of silica may predispose to or initiate the development of SLE. The detection of SLE-typical antibodies in quartz dust-exposed miners may indicate a higher risk for the development of systemic autoimmune disease.Antimalarial drugs are effective disease modifying agents in RA with a low incidence of serious toxic effects. Recently, combinations of second-line agents have been used in RA in attempts to treat patients with no response to a number of single agents, or suboptimal response to a single agent. Combinations of drugs have been selected for maximum efficacy and minimum toxicity, but clinical trials are difficult to design and interpret. In particular, ensuring adequate power to detect small differences in response poses a major problem. Antimalarials are an attractive choice for combination therapy due to their efficacy, mechanisms of action and toxicity profile. In this review, the evidence for the use of antimalarials in combination in RA is examined. No advantage has been shown in combining antimalarials with gold, penicillamine or sulphasalazine compared with monotherapeutic regimens. There is some evidence to suggest a beneficial combination of antimalarials with methotrexate, but this is as yet inconclusive. Open non-randomised uncontrolled studies have shown that antimalarials combined with cytotoxic agents are effective but highly toxic. The authors conclude that there is little good evidence to support the introduction of combination second-line drug therapy for RA into widespread therapeutic use.

Profiling of rheumatoid arthritis associated autoantibodies

An increasing number of rheumatoid arthritis (RA)-associated autoantibodies (AAB) are available for AAB profiling that may improve the early diagnosis of RA and provide prognostic and, probably theranostic information. To select AAB specificities for optimal AAB combinations, known AAB should be evaluated with standardized methods by means of standardized study designs and subjected to statistical analysis. Profiling of anti-citrullinated peptide/protein antibodies (ACPA), anti-A2/RA-33 antibodies, and rheumatoid factors (RF) IgM and IgA improves the serologic diagnosis of RA using cut-offs corresponding to 98% specificity for each parameter included. Currently, the combination of anti-CCP antibodies with RF IgM and RF IgA is recommended either in parallel or by stepwise determination. Because anti-CCP antibody demonstrated the best diagnostic performance for profiling, this AAB should be used for first-line screening. The main advantage of AAB profiling is the increased sensitivity for RA diagnosis. Additional benefits of AAB profiles comprise the increased diagnostic specificity of particular AAB combinations (e.g., ACPA plus RF or RF IgM plus RF IgA) and their possible association with disease development and/or therapy response. The inclusion of novel RA-associated AAB (RAAB) and use of clinically evaluated multiplex assays may facilitate the use of profiling in diagnostic routine laboratories.

Autoantibody Detection Using Indirect Immunofluorescence on HEp-2 Cells

The detection of autoantibodies is an important element in the diagnosis and monitoring of disease progression in patients with autoimmune diseases. In laboratory diagnostic tests for connective tissue and autoimmune liver diseases, indirect immunofluorescence on HEp‐2 cells plays a central role in a multistage diagnostic process. Despite the high quality of diagnostics, findings at different laboratories can differ considerably due to a lack of standardization, as well as subjective factors.

Asialoglycoprotein receptor (ASGPR) as target autoantigen in liver autoimmunity: lost and found.

Asialoglycoprotein receptor (ASGPR) has attracted the attention of liver immunologists for many years. This liver-specific lectin was found to be a major B and T cell autoantigenic target in patients with autoimmune liver diseases, and in particular in autoimmune hepatitis (AIH). This review discusses the biological significance of ASGPR and its relevance to the pathogenesis of autoimmune and virus-triggered liver diseases. We also discuss emerging data on the diagnostic and clinical relevance of anti-ASGPR antibodies in light of recent reports based on commercially available anti-ASGPR enzyme-linked immunosorbent assays. Finally, we critically revisit the data reporting on disease-specific cellular immune responses against ASGPR and their relevance in relation to the pathogenesis of AIH.