[The baseline assessment of the German National Cohort (NAKO Gesundheitsstudie): participation in the examination modules, quality assurance, and the use of secondary data]. / Die Basiserhebung der NAKO Gesundheitsstudie: Teilnahme an den Untersuchungsmodulen, Qualitätssicherung und Nutzung von Sekundärdaten.

BACKGROUND: The German National Cohort (NAKO) is an interdisciplinary health study aimed at elucidating causes for common chronic diseases and detecting their preclinical stages. This article provides an overview of design, methods, participation in the examinations, and their quality assurance based on the midterm baseline dataset (MBD) of the recruitment. METHODS: More than 200,000 women and men aged 20-69 years derived from random samples of the German general population were recruited in 18 study centers (2014-2019). The data collection comprised physical examinations, standardized interviews and questionnaires, and the collection of biomedical samples for all participants (level 1). At least 20% of all participants received additional in-depth examinations (level 2), and 30,000 received whole-body magnet resonance imaging (MRI). Additional information will be collected through secondary data sources such as medical registries, health insurances, and pension funds. This overview is based on the MBD, which included 101,839 participants, of whom 11,371 received an MRI. RESULTS: The mean response proportion was 18%. The participation in the examinations was high with most of the modules performed by over 95%. Among MRI participants, 96% completed all 12 MRI sequences. More than 90% of the participants agreed to the use of complementary secondary and registry data. DISCUSSION: Individuals selected for the NAKO were willing to participate in all examinations despite the time-consuming program. The NAKO provides a central resource for population-based epidemiologic research and will contribute to developing innovative strategies for prevention, screening and prediction of chronic diseases.

Cholesteryl esters stabilize human CD1c conformations for recognition by self-reactive T cells.

Cluster of differentiation 1c (CD1c)-dependent self-reactive T cells are abundant in human blood, but self-antigens presented by CD1c to the T-cell receptors of these cells are poorly understood. Here we present a crystal structure of CD1c determined at 2.4 Å revealing an extended ligand binding potential of the antigen groove and a substantially different conformation compared with known CD1c structures. Computational simulations exploring different occupancy states of the groove reenacted these different CD1c conformations and suggested cholesteryl esters (CE) and acylated steryl glycosides (ASG) as new ligand classes for CD1c. Confirming this, we show that binding of CE and ASG to CD1c enables the binding of human CD1c self-reactive T-cell receptors. Hence, human CD1c adopts different conformations dependent on ligand occupancy of its groove, with CE and ASG stabilizing CD1c conformations that provide a footprint for binding of CD1c self-reactive T-cell receptors.

Acylated cholesteryl galactosides are ubiquitous glycolipid antigens among Borrelia burgdorferi sensu lato.

Lyme disease (LD) is the most common tick-borne disease in the Northern hemisphere. It is caused by Borrelia burgdorferi sensu lato, in particular, B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii. However, other genospecies have been implicated as causative factors of LD as well. Borrelia burgdorferi exhibits numerous immunogenic lipoproteins, but due to strong heterogeneity, the use of these proteins for serodiagnosis and vaccination is hampered. We and others have identified acylated cholesteryl galactosides (ACGal) as a novel glycolipid present in B. burgdorferi sensu stricto, B. afzelii, and B. garinii. ACGal is a strong antigen and the majority of patients display anti-ACGal antibodies in the chronic stages of LD. However, it is unknown whether ACGal is present in other presumably pathogenic B. burgdorferi genospecies. Therefore, we performed an analysis of the total lipid extracts of a wide spectrum of genospecies of B. burgdorferi sensu lato using thin-layer chromatography as well as Western blot and dot-blot assays. We show that ACGal is present in substantial quantities in all B. burgdorferi genospecies tested. Therefore, this molecule might improve the serological detection of rarely pathogenic genospecies, and may be used as a protective vaccine regardless of the prevailing genospecies.

Chemoenzymatic synthesis of a glycolipid library and elucidation of the antigenic epitope for construction of a vaccine against Lyme disease.

Lyme disease (LD) is the most common tick-borne disease in Europe, North America, and Asia. The etiologic agents of LD are spirochetes of the group Borrelia burgdorferi sensu lato, which possess a lipid content of 25-30% of the dry weight. The major glycolipid cholesteryl 6-O-acyl-beta-D-galactopyranoside (ACGal), present in B. burgdorferi sensu stricto, B. afzelii, and B. garinii, is a specific and highly prevalent antigen frequently recognized by antibodies in late-stage LD. Here we report a convenient route for the chemical synthesis of ACGal by employing a combination of chemical synthesis steps with enzymatic transformations. This synthesized molecule was compared with bacterial extracts by immunoblots with patient sera, confirming the preserved antigenicity. Next, a glycolipid library derived from the native molecules with variations in the fatty acyl moiety and derivatives in which the cholesterol has been replaced was designed and synthesized. The chemical structures were confirmed by 1D and 2D NMR spectroscopy and mass spectrometry. The native and synthetic glycolipids were utilized in immunoblots to determine the epitope recognized by antibodies in patient sera. By this method we could demonstrate that galactose, cholesterol, and a fatty acid with a minimal chain length of four carbon atoms comprises the essential structure for recognition by antibodies. Finally, this finding allowed the synthesis of a functionalized ACGal with an omega-mercapto group at the fatty acid and a facile protection and deprotection strategy. This antigenic hapten can be conjugated to a carrier protein to effect immunization against Lyme disease.

Acylated cholesteryl galactosides are specific antigens of borrelia causing lyme disease and frequently induce antibodies in late stages of disease.

Borrelia burgdorferi sensu lato is the causative agent of Lyme disease (LD), an infectious disease occurring in North America, Europe, and Asia in different clinical stages. B. burgdorferi sensu lato encompasses at least 12 species, with B. burgdorferi sensu stricto, B. garinii, and B. afzelii being of highest clinical importance. Immunologic testing for LD as well as recent vaccination strategies exclusively refer to proteinaceous antigens. However, B. burgdorferi sensu stricto exhibits glycolipid antigens, including 6-O-acylated cholesteryl beta-D-galactopyranoside (ACGal), and first the data indicated that this compound may act as an immunogen. Here we investigated whether B. garinii and B. afzelii also possess this antigen, and whether antibodies directed against these compounds are abundant among patients suffering from different stages of LD. Gas-liquid chromatography/mass spectroscopy and NMR spectroscopy showed that both B. garinii and B. afzelii exhibit ACGal in high quantities. In contrast, B. hermsii causing relapsing fever features 6-O-acylated cholesteryl beta-D-glucopyranoside (ACGlc). Sera derived from patients diagnosed for LD contained antibodies against ACGal, with 80% of patients suffering from late stage disease exhibiting this feature. Antibodies reacted with ACGal from all three B. burgdorferi species tested, but not with ACGlc from B. hermsii. These data show that ACGal is present in all clinically important B. burgdorferi species, and that specific antibodies against this compound are frequently found during LD. ACGal may thus be an interesting tool for improving diagnostics as well as for novel vaccination strategies.

Immune responses induced by spirochetal outer membrane lipoproteins and glycolipids.

The class of Spirochetes comprises a wide array of clinically important pathogens, including Treponema pallidum causing syphilis as well as Borrelia burgdorferi, the agent of Lyme disease (LD). Diseases caused by spirochetes are characterized by specific sequelae of host reactions, and also by characteristic antibody response patterns. Over the last decades, research on the interaction of spirochetes with the host's immune system had a strong emphasis on outer membrane lipoproteins. In fact, these structures have been convincingly shown to activate immune cells via CD14 and Toll-like receptor (TLR)-2, and recent data also indicate an interaction with lipopolysaccharide (LPS)-binding protein (LBP). In particular, the interaction of B. burgdorferi with TLR-2 could not only be demonstrated in mice, but was also supported by data showing that genetic variants of TLR-2 in humans influenced the clinical course of LD. However, there is increasing evidence that next to lipoproteins, glycolipids may also play an important role in responses of the immune system towards spirochetes. Diacylglycerol-containing glycolipids exhibiting similarities with lipoteichoic acid (LTA) of Gram-positive bacteria have been demonstrated in various Treponema species, whereas LPS-like glycolipids have been shown to be present in Leptospira. Treponema glycolipids, comparably to lipoproteins and LTA, interact with LBP, CD14 and TLRs. In contrast, complex glycolipids of high molecular weight could not be demonstrated in Borrelia, whereas these bacteria exhibit a number of unique low molecular weight glycolipids. Some of these glycolipids cause strong immediate immune responses, while others appear to be potent antigens for induction of an adaptive immune response. This review summarizes data obtained so far on amphiphilic and hydrophobic molecules from spirochetes regarding structure and influence on innate as well as adaptive immune responses.