Thorsten Krieger

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.

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.

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.

Autoantikörpernachweis mittels indirekter Immunfluoreszenz an HEp-2-Zellen

Systemic autoimmune diseases are characterized by the presence of antinuclear autoantibodies (ANA). Diluted patient sera are typically used to screen for the presence of ANA by immunfluorescence microscopy with fixed HEp-2 cells. Despite high-quality test kits, reports of different laboratories frequently present controversial results. This article recommends unified processing and interpretation of HEp-2 based screening for autoantibodies. Suggestions are made for the selection of high-quality test kits, optimized processing and diagnostic procedures. In addition to a relevant clinical diagnosis and an experienced laboratory specialist, the following procedure is highly recommended to achieve good laboratory practice: Initial HEp-2 based screening by indirect immunofluorescence, starting with a 1:80 serum dilution, and evaluation in a bright fluorescence microscope, pathological values from a titer of 1:160 upwards, internal quality checks and unified interpretation. We aim to improve diagnosis and care of patients with autoimmune diseases as a central focus of the European Autoimmunity Standardization Initiative (EASI).

Cloning and characterization of an adenoviral vector for highly efficient and doxycycline – suppressible expression of bioactive human single – chain interleukin 12 in colon cancer

Background Interleukin-12 (IL-12) is well characterized to induce cellular antitumoral immunity by activation of NK-cells and T-lymphocytes. However, systemic administration of recombinant human IL-12 resulted in severe toxicity without perceptible therapeutic benefit. Even though intratumoral expression of IL-12 leads to tumor regression and long-term survival in a variety of animal models, clinical trials have not yet shown a significant therapeutic benefit. One major obstacle in the treatment with IL-12 is to overcome the relatively low expression of the therapeutic gene without compromising the safety of such an approach. Our objective was to generate an adenoviral vector system enabling the regulated expression of very high levels of bioactive, human IL-12. Results High gene expression was obtained utilizing the VP16 herpes simplex transactivator. Strong regulation of gene expression was realized by fusion of the VP16 to a tetracycline repressor with binding of the fusion protein to a flanking tetracycline operator and further enhanced by auto-regulated expression of its fusion gene within a bicistronic promoter construct. Infection of human colon cancer cells (HT29) at a multiplicity of infection (m.o.i.) of 10 resulted in the production of up to 8000 ng/106 cells in 48 h, thus exceeding any published vector system so far. Doxycycline concentrations as low as 30 ng/ml resulted in up to 5000-fold suppression, enabling significant reduction of gene expression in a possible clinical setting. Bioactivity of the human single-chain IL-12 was similar to purified human heterodimeric IL-12. Frozen sections of human colon cancer showed high expression of the coxsackie adenovirus receptor with significant production of human single chain IL-12 in colon cancer biopsies after infection with 3*107 p.f.u. Ad.3r-scIL12. Doxycycline mediated suppression of gene expression was up to 9000-fold in the infected colon cancer tissue. Conclusion VP16 transactivator-mediated and doxycycline-regulated expression of the human interleukin-12 gene enables highly efficient and tightly controlled cytokine expression in human cancer. These data illustrate the potential of the described adenoviral vector system for the safe and superior expression of therapeutic genes in the treatment of colorectal cancer and other malignancies.BackgroundInterleukin-12 (IL-12) is well characterized to induce cellular antitumoral immunity by activation of NK-cells and T-lymphocytes. However, systemic administration of recombinant human IL-12 resulted in severe toxicity without perceptible therapeutic benefit. Even though intratumoral expression of IL-12 leads to tumor regression and long-term survival in a variety of animal models, clinical trials have not yet shown a significant therapeutic benefit. One major obstacle in the treatment with IL-12 is to overcome the relatively low expression of the therapeutic gene without compromising the safety of such an approach. Our objective was to generate an adenoviral vector system enabling the regulated expression of very high levels of bioactive, human IL-12.ResultsHigh gene expression was obtained utilizing the VP16 herpes simplex transactivator. Strong regulation of gene expression was realized by fusion of the VP16 to a tetracycline repressor with binding of the fusion protein to a flanking tetracycline operator and further enhanced by auto-regulated expression of its fusion gene within a bicistronic promoter construct. Infection of human colon cancer cells (HT29) at a multiplicity of infection (m.o.i.) of 10 resulted in the production of up to 8000 ng/106 cells in 48 h, thus exceeding any published vector system so far. Doxycycline concentrations as low as 30 ng/ml resulted in up to 5000-fold suppression, enabling significant reduction of gene expression in a possible clinical setting. Bioactivity of the human single-chain IL-12 was similar to purified human heterodimeric IL-12. Frozen sections of human colon cancer showed high expression of the coxsackie adenovirus receptor with significant production of human single chain IL-12 in colon cancer biopsies after infection with 3*107 p.f.u. Ad.3r-scIL12. Doxycycline mediated suppression of gene expression was up to 9000-fold in the infected colon cancer tissue.ConclusionVP16 transactivator-mediated and doxycycline-regulated expression of the human interleukin-12 gene enables highly efficient and tightly controlled cytokine expression in human cancer. These data illustrate the potential of the described adenoviral vector system for the safe and superior expression of therapeutic genes in the treatment of colorectal cancer and other malignancies.

Standardization of autoimmune diagnostics in Germany: activities of the German group in the European Autoimmune Standardization Initiative.

Abstract:  The German Regional Group of EASI was established during the annual Meeting of the German Society of Immunology in Kiel in September 2005. Since this initial informative meeting, an active core group of about a dozen rheumatologists, immunologists, and laboratory specialists has been generating starter projects. In general, these projects do focus on clinically associated diagnostic questions, and do integrate a variety of specialists with profound knowledge in several related subjects. The aims of the German EASI group are to contribute to the definition of standards and to improve patient care. Therefore, the group is establishing guidelines for the diagnosis of autoimmune diseases, to standardize and improve their quality, combining the experience of clinical and laboratory specialists. The diagnostic activities focus currently on systemic lupus erythematosus (SLE) and on rheumatoid arthritis. These activities include laboratory investigations and diagnosis through clinical manifestations. Standardized diagnostics cannot be based solely on vague symptoms and positive laboratory tests. In laboratory diagnostics, standardization and implementation of objective methods for the detection of autoantibodies has been identified as a central challenge. Here, immune fluorescence techniques and the evaluation of RibP are used as first parameters that could improve SLE diagnostics. Furthermore, guidelines and proposals from scientific medical organizations, and in particular from other national EASI groups will be adapted to the German health system. A cornerstone of implementation is the identification and logistic preparation of existing serum banks, the definition of gaps that should be bridged, and, particularly, the definition and collection of adequate control groups. Through these measures, the German EASI group will provide a standardized diagnostic model of autoimmune disorders throughout Europe starting in the field of rheumatology. Diagnostics may become more rational, efficient, faster, and cost‐efficient. Patients with autoimmune rheumatic disorders will profit from receiving an earlier and more accurate diagnosis, which again will allow earlier therapeutic intervention and lead to a better long‐term clinical outcome.

Peripheral blood biomarkers for the characterization of alloimmune reactivity after pediatric liver transplantation

Individualization of immunosuppressive medications is an important objective in transplantation medicine. Reliable biomarkers to distinguish between patients dependent from intensive immunosuppressive therapy and those where therapy can be minimized among pediatric transplant recipients receiving immunosuppressive medications are still not established. We evaluated the potential of cross‐sectional quantification of regulatory T cells, lymphocyte subsets, and cytokine concentrations as biomarkers in 60 pediatric liver transplant recipients with AR, CR, or normal graft function and in 11 non‐transplanted patients. Transplant recipients presenting with AR had significantly higher CD8+ T‐cell counts, significantly higher concentrations of IL‐2, and increased levels of IFN‐γ compared with asymptomatic patients or controls. Regulatory T‐cell numbers did not differ between children with rejection and children with good graft function. A tendency toward increased concentrations of IL‐4 and TGF‐β was detected in transplant recipients with good graft function. Cross‐sectional parameters of peripheral regulatory T cells in pediatric liver transplant recipients do not seem to be valuable biomarkers for individualizing immunosuppressive therapy prior to the weaning process. Lymphocyte subsets, IL‐2, IFN‐γ, IL‐4, and TGF‐β serum concentrations may be helpful to identify children in whom immunosuppression can be reduced or discontinued.

Neutrophil Extracellular Traps Contain Selected Antigens of Anti-Neutrophil Cytoplasmic Antibodies

Neutrophil extracellular traps (NETs) are chromatin filaments decorated with enzymes from neutrophil cytoplasmic granules. Anti-neutrophil cytoplasmic antibodies (ANCAs) bind to enzymes from neutrophil cytoplasmic granules and are biomarkers for the diagnosis of systemic vasculitides. ANCA diagnostics are based on indirect immunofluorescence (IIF) of ethanol-fixed neutrophils. IIF shows a cytoplasmic staining pattern (C-ANCA) due to autoantibodies against proteinase 3 (PR3) or a perinuclear staining pattern (P-ANCA) due to autoantibodies against myeloperoxidase (MPO). The distinct ANCA-staining patterns are an artifact of ethanol fixation. Here, we tested NETs as a substrate for the detection of ANCAs in human sera. We observed that P-ANCAs specifically stained NETs, while C-ANCAs targeted the cell bodies of netting neutrophils. The distinct ANCA-staining patterns were caused by the presence of MPO, but not PR3, in NETs. Using NETs as a substrate for IIF, we characterized ANCAs in sera of patients with ANCA-associated vasculitis (AAV). Furthermore, we inhibited serine proteases by diisopropylfluorophosphate to prevent chromatin unfolding and the release of NETs and thus generated neutrophils with MPO-positive nuclei and PR3-positive cytoplasm, which resembled the appearance of ethanol-fixed neutrophils. In conclusion, our data suggest that NETs are selectively loaded with antigens recognized by P-ANCAs, and netting neutrophils provide a physiological substrate for ANCA detection in patients with AAV.

BCL11B mutations in patients affected by a neurodevelopmental disorder with reduced type 2 innate lymphoid cells

The transcription factor BCL11B is essential for development of the nervous and the immune system, and Bcl11b deficiency results in structural brain defects, reduced learning capacity, and impaired immune cell development in mice. However, the precise role of BCL11B in humans is largely unexplored, except for a single patient with a BCL11B missense mutation, affected by multisystem anomalies and profound immune deficiency. Using massively parallel sequencing we identified 13 patients bearing heterozygous germline alterations in BCL11B. Notably, all of them are affected by global developmental delay with speech impairment and intellectual disability; however, none displayed overt clinical signs of immune deficiency. Six frameshift mutations, two nonsense mutations, one missense mutation, and two chromosomal rearrangements resulting in diminished BCL11B expression, arose de novo. A further frameshift mutation was transmitted from a similarly affected mother. Interestingly, the most severely affected patient harbours a missense mutation within a zinc-finger domain of BCL11B, probably affecting the DNA-binding structural interface, similar to the recently published patient. Furthermore, the most C-terminally located premature termination codon mutation fails to rescue the progenitor cell proliferation defect in hippocampal slice cultures from Bcl11b-deficient mice. Concerning the role of BCL11B in the immune system, extensive immune phenotyping of our patients revealed alterations in the T cell compartment and lack of peripheral type 2 innate lymphoid cells (ILC2s), consistent with the findings described in Bcl11b-deficient mice. Unsupervised analysis of 102 T lymphocyte subpopulations showed that the patients clearly cluster apart from healthy children, further supporting the common aetiology of the disorder. Taken together, we show here that mutations leading either to BCL11B haploinsufficiency or to a truncated BCL11B protein clinically cause a non-syndromic neurodevelopmental delay. In addition, we suggest that missense mutations affecting specific sites within zinc-finger domains might result in distinct and more severe clinical outcomes.

Comparison of modern analyzers for the detection of antiphospholipid antibodies in patients with SLE

Abstract Background: The anti-cardiolipin and β2-glycoprotein I antibodies represent the important diagnostic parameters in routine autoimmune screening and represent the important diagnostic criteria for systemic lupus erythematosus (SLE). In this study, five different automated, semiautomated, and manual immunoassays detecting IgG/IgM anti-cardiolipin and anti-β2-glycoprotein I antibodies were tested. Methods: A total of 43 samples from the patients with SLE diagnosed in the rheumatology outpatient centers in Germany were included in this study. Results: For anticardiolipin and anti-β2-glycoprotein I, considerable differences in the percentage of the positive results were found between all the five methods, and the itemization of all the positive test results reached only a poor accordance. These findings were confirmed by Cohen’s κ coefficients. Conclusions: We found a moderate to poor accordance between the five different modern test systems for anticardiolipin and anti-β2-glycoprotein I antibodies. Such discrepancies can result in the clinical misinterpretation of data and may lead to wrong diagnoses. Therefore, further standardization of the tests for the antiphospholipid antibodies (APAs) is required. Zusammenfassung Hintergrund: Anti-Cardiolipin und β2-Glykoprotein I Antikörper präsentieren wichtige Parameter in der Routinediagnostik von Autoimmunerkrankungen und stellen ein wesentliches diagnostisches Kriterium für die Diagnose eines systemischen Lupus erythematodes (SLE) dar. In dieser Studie wurden erstmals alle gängigen modernen voll- und teilautomatisierten Analysesysteme hinsichtlich der Bestimmung von anti-Cardiolipin und β2-Glykoprotein I-Antikörpern miteinander verglichen. Methoden: Es wurden 43 Proben von Patienten mit diagnostiziertem systemischen Lupus erythematodes untersucht. Ergebnisse: Sowohl bei der Bestimmung von anti-Cardiolipin als auch von β2-Glykoprotein I-Antikörpern zeigten sich signifikante Unterschiede zwischen den verschiedenen Verfahren. Statistisch konnte dies durch Einzelbetrachtung der Ergebnisse mittels des Cohens Kappa-Koeffizienten verdeutlicht werden. Schlussfolgerung: Unsere Ergebnisse zeigen eine schwache bis fehlende Übereinstimmung der analytischen Resultate bei der Bestimmung von anti-Cardiolipin und von β2-Glykoprotein I-Antikörpern zwischen den aktuellen teil-/ vollautomatisierten Analyzern. Wesentlich für diese Beurteilung war dabei die Betrachtung der Einzelergebnisse und nicht die der gemittelten Gesamtheit. Abweichungen dieses Ausmaßes können in einer klinischen Fehldiagnose und fehlerhaften Therapie resultieren. Eine weitere Standardisierung ist erforderlich.