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.HINTERGRUND: Die NAKO Gesundheitsstudie ist ein bundesweites interdisziplinäres Forschungsvorhaben mit dem Ziel, die Ursachen für chronische Krankheiten und deren vorklinische Stadien zu untersuchen. Der Artikel gibt einen Überblick über das Studiendesign, die Methoden, die Teilnahme an den Untersuchungen und ihre Qualitätssicherung zur Halbzeit der Basiserhebung. METHODEN: Für die Basiserhebung wurden mehr als 200.000 Frauen und Männer im Alter von 20–69 Jahren aus Zufallsstichproben der Allgemeinbevölkerung in 18 Studienzentren rekrutiert (2014–2019). Die Basiserhebung beinhaltet Untersuchungen, Befragungen und Biomaterialien für alle Teilnehmerinnen und Teilnehmer (Level 1), ein erweitertes Programm für mindestens 20 % (Level 2) und eine Magnetresonanztomografie (MRT) für 30.000 Teilnehmerinnen und Teilnehmer. Sekundär- und Registerdaten werden über Krankheitsregister, Kranken- und Rentenversicherungen erhoben. Die Auswertung bezieht die Datenbasis zur Halbzeit der Basiserhebung mit 101.839 Teilnehmerinnen und Teilnehmern ein, davon 11.371 mit einer MRT-Untersuchung. ERGEBNISSE: Die mittlere Responsequote zur Halbzeit betrug insgesamt 18 %. Die Teilnahme an den Untersuchungen lag überwiegend bei mehr als 95 %. Bei 96 % der MRT-Teilnehmerinnen und Teilnehmer konnten alle 12 MRT-Sequenzen vollständig durchgeführt werden. Der Erschließung und wissenschaftlichen Nutzung ergänzender Sekundär- und Registerdaten stimmten mehr als 90 % der Teilnehmerinnen und Teilnehmer zu. DISKUSSION: Die Bereitschaft, möglichst alle Untersuchungsmodule durchzuführen, war trotz des zeitlichen Aufwandes außerordentlich hoch. Dadurch wird die NAKO zu einer zentralen Ressource für die epidemiologische Forschung in Deutschland. Sie wird es ermöglichen, neue Strategien zur Früherkennung, Vorhersage und Primärprävention chronischer Krankheiten zu entwickeln

Framework and baseline examination of the German National Cohort (NAKO)

The German National Cohort (NAKO) is a multidisciplinary, population-based prospective cohort study that aims to investigate the causes of widespread diseases, identify risk factors and improve early detection and prevention of disease. Specifically, NAKO is designed to identify novel and better characterize established risk and protection factors for the development of cardiovascular diseases, cancer, diabetes, neurodegenerative and psychiatric diseases, musculoskeletal diseases, respiratory and infectious diseases in a random sample of the general population. Between 2014 and 2019, a total of 205,415 men and women aged 19–74 years were recruited and examined in 18 study centres in Germany. The baseline assessment included a face-to-face interview, self-administered questionnaires and a wide range of biomedical examinations. Biomaterials were collected from all participants including serum, EDTA plasma, buffy coats, RNA and erythrocytes, urine, saliva, nasal swabs and stool. In 56,971 participants, an intensified examination programme was implemented. Whole-body 3T magnetic resonance imaging was performed in 30,861 participants on dedicated scanners. NAKO collects follow-up information on incident diseases through a combination of active follow-up using self-report via written questionnaires at 2–3 year intervals and passive follow-up via record linkages. All study participants are invited for re-examinations at the study centres in 4–5 year intervals. Thereby, longitudinal information on changes in risk factor profiles and in vascular, cardiac, metabolic, neurocognitive, pulmonary and sensory function is collected. NAKO is a major resource for population-based epidemiology to identify new and tailored strategies for early detection, prediction, prevention and treatment of major diseases for the next 30 years. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10654-022-00890-5

Investigations on the glycolipids of Borrelia burgdorferi, the etiologic agent of Lyme Disease, and its application in serodiagnostics as well as a vaccine

Lyme-Borreliose (LB) ist eine Infektionskrankheit, deren Erreger einige Bakterienarten aus der taxonomischen Gruppe Borrelia burgdorferi sensu lato ("im weiteren Sinn") sind. Die Diagnose erfolgt aufgrund der klinischen Manifestationen und einer bestätigenden Serologie. Die Serodiagnostik und die Impfstoffentwicklung werden durch die Heterogenität der Proteinantigene zwischen und teilweise innerhalb der Bakterienarten vor große Herausforderungen gestellt. Ein Impfstoff gegen LB ist weder aktuell verfügbar, noch in Aussicht. Das Ziel dieser Arbeit war es, die Glycolipide von B. burgdorferi sensu lato eingehend zu analysieren und auf ihre Einsetzbarkeit für beide medizinischen Anwendungen hin zu untersuchen. Es wird dargelegt, dass die verschiedenen Arten unabhängig von ihrer Pathogenität die gleichen Glycolipide aufweisen und diese etwa 13 % der Borrelientrockenmasse ausmachen. Die Glycolipide der drei klinisch wichtigsten Pathogene wurden isoliert, chromatographisch aufgereinigt, quantifiziert und chemisch mittels GLC/MS, NMR und MALDI-TOF analysiert. Die dominierende Struktur ist Cholesteryl-6-O-acyl-β-D-galactopyranosid (ACGal), das fast die Hälfte der Glycolipide und damit 5-6 % des Borreliengewichtes ausmacht. Jeweils etwa ein Viertel stellen Cholesteryl-β-D-galactopyranosid (CGal) sowie Mono-α-D-galactosyldiacylglycerol (MGalD) und ein kleiner Anteil ist Cholesteryl-β-D-glucopyranosid (CGlc). Sowohl ACGal als auch CGal sind innerhalb von Bakterien und Tieren einmalige Strukturen. Experimente mit Zellkulturen zeigten, dass keines der Glycolipide von den Toll-like-Rezeptoren des angeborenen Immunsystems erkannt wird. Eine Untersuchung der Gesamtlipide mit LB-Patientenseren identifizierte dagegen ACGal und MGalD als Antigene des erworbenen Immunsystems. Die Analyse dieser beiden isolierten Antigene mit 174 LB-Patientenseren mittels Dot-Blots oder Line-Blots ergab, dass im Spätstadium der LB 83 % bzw. 63 % der Seren Antikörper gegen ACGal mit Titern bis 1:32.000 aufwiesen. Der eng verwandte Erreger des Rückfallfiebers, Borrelia hermsii, verfügt dagegen statt ACGal über Cholesteryl-6-O-acyl-β-D-glucopyranosid, das von LB-Seren nicht erkannt wird. Ebenso ist die Spezifität von ACGal bei oft kreuzreagierenden Seren anderer Erkrankungen sehr gut. ACGal kann daher zusätzlich zu den Proteinantigenen zur Verbesserung von kommerziellen Borrelien-Serodiagnostik-Kits eingesetzt werden und wird von einer Firma bereits verwendet. Um die Eignung von ACGal als Impfstoff zu untersuchen, wurde ein effizienter chemisch-enzymatischer Syntheseweg konzipiert und erprobt. Dabei wurde CGal zunächst mittels der Trichloracetimidatmethode dargestellt und dann mittels einer Lipase regioselektiv zum ACGal acyliert. Diese Vorgehensweise wurde zum Aufbau einer Glycolipidbibliothek mit diversen ACGal-Derivaten verwendet, die zur Bestimmung des Epitops der Anti-ACGal-Antikörper in den LB-Patientenseren diente. Die Kenntnis des antigenen Epitops ermöglichte wiederum die Synthese eines funktionalisierten ACGal. Für den dabei verwendeten ω-Mercaptoacyl-Rest wurde eine einfache Schützung und Entschützung mit der flüchtigen Thioethylgruppe etabliert. Zur Konjugation des hydrophoben, funktionalisierten ACGal an hydrophile Trägerproteine wurde eine neue Methode entwickelt, die reproduzierbar hohe Beladungsgrade erzielt. Mit diesen immunogenen ACGal- Konjugaten wurden Kaninchen erfolgreich immunisiert und die generierten Antikörper mittels Immunoblots charakterisiert. Die bestimmten Antikörpertiter unterschieden sich zwischen den ACGal-Derivaten und erreichen Werte von 1:128.000 für natives ACGal und 1:512.000 für kurzkettige, artifizielle ACGal- Derivate, wobei das Epitop mit dem der humanen Antikörper übereinstimmt. Mittels Immunofluoreszenz wurde gezeigt, dass die Antiseren an intakte Borrelien binden und das Epitop somit für Antikörper zugänglich scheint. Nicht mehr untersucht wurde, ob die Antikörper die Borrelien opsonieren oder sogar abtöten können. In noch ausstehenden Tierversuchen muss geklärt werden, ob eine Impfung mit ACGal-Konjugat vor einer Infektion mit B. burgdorferi schützen kann.Lyme Disease (LD) is an infectious disease caused by several bacterial species of the taxonomic group of Borrelia burgdorferi sensu lato ("in a wider sense"). The diagnosis is based on clinical manifestations and confirmed by serology. However, serodiagnostics as well as the development of a vaccine are hampered by the heterogeneity of the protein antigens between or even within the bacterial species. A vaccine is neither available today nor foreseeable. The aim of this work was to analyze and evaluate the glycolipids of B. burgdorferi sensu lato for both medical applications. It is shown that different species possess identical glycolipids independent of their pathogenicity accounting for approx. 13 % of the cellular dry weight. The glycolipids of the clinically most relevant species were isolated, separated by column chromatography, quantified and analyzed by GLC/MS, NMR, and MALDI- TOF. The dominant structure is cholesteryl-6-O-acyl-β-D-galactopyranoside (ACGal) representing nearly half of the glycolipids and therefore 5-6 % of the borrelial mass. About one quarter of the glycolipids are composed of cholesteryl-β-D-galactopyranoside (CGal) and mono-α-D-galactosyldiacylglycerol (MGalD), and a minor proportion constitutes cholesteryl-β-D-glucopyranoside (CGlc). ACGal as well as CGal are unique structures within the bacteria and the animal kingdom. Experiments with cell cultures revealed that none of the glycolipids is recognized by toll-like receptors of the innate immune system. However, screening of the total lipids with LD patient sera identified ACGal and MGalD as antigens of the adaptive immune system. Subsequent analyzes of both antigens with 174 LD patient sera using dot-blots or line-blots demonstrated the highest seroprevalence in the late stage of disease. Then 83, and 63 % respectively of the sera contained antibodies against ACGal with titers up to 1:32,000. On the other hand the closely related causative agent of tick-borne relapsing fever, Borrelia hermsii, instead of ACGal contains cholesteryl-6-O-acyl-β-D-glucopyranoside that is not recognized by LD sera. Also, the specificity of ACGal for cross-reacting sera of patients with other diseases is excellent. Taken together, ACGal can be employed in addition to proteinacious antigens to improve commercial Borrelia serodiagnostics kits and one company is currently pursuing this. To assess the use of ACGal as a vaccine, an efficient chemo-enzymatic synthesis route was established. First, CGal was prepared by the trichloroacetimidate method. Next it was acylated regioselectively to ACGal by lipase. This approach was utilized to build a glycolipid library consisting of diverse ACGal derivatives to determine the epitope of the anti-ACGal antibodies in LD patient sera. Knowledge of the antigenic epitope enabled the synthesis of a functionalized ACGal molecule. For the employed ω-mercaptoacyl residue a simple protection and deprotection strategy was developed using the evaporable thioethyl protecting group. This allows the conjugation of ACGal to immunogenic carrier proteins. To achieve high conjugation grades in a reproducible fashion a new method was designed to bring these hydrophobic and hydrophilic molecules into reaction. These ACGal conjugates were used to successfully immunize rabbits. The antibodies generated were characterized by different immunoblotting methods. The determined antibody titers varied between the ACGal derivatives and reached values of 1:128,000 for native ACGal and 1:512,000 for artificial short-chain ACGal derivatives. However, the epitope coincided with the one detected by human antibodies. Applying immunofluorescence it could be shown that the antisera bound to whole Borrelia and that the epitope seems to be accessible to the antibodies. It was not examined whether antibodies opsonize Borrelia or are capable of killing them. Furthermore, it remains to be investigated in animal experiments whether a vaccination with an ACGal conjugate is able to prevent an infection by B. burgdorferi

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

CD1c antigen presentation

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 unknown. 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 to known CD1c structures. Computational simulations exploring different occupancy states of the groove re-enacted 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 stabilising CD1c conformations that provide a footprint for binding of CD1c self-reactive T cell receptors

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 β-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 β-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

[The baseline assessment of the German National Cohort (NAKO Gesundheitsstudie): participation in the examination modules, quality assurance, and the use of secondary data].

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

Framework and baseline examination of the German National Cohort (NAKO)

The German National Cohort (NAKO) is a multidisciplinary, population-based prospective cohort study that aims to investigate the causes of widespread diseases, identify risk factors and improve early detection and prevention of disease. Specifically, NAKO is designed to identify novel and better characterize established risk and protection factors for the development of cardiovascular diseases, cancer, diabetes, neurodegenerative and psychiatric diseases, musculoskeletal diseases, respiratory and infectious diseases in a random sample of the general population. Between 2014 and 2019, a total of 205,415 men and women aged 19–74 years were recruited and examined in 18 study centres in Germany. The baseline assessment included a face-to-face interview, self-administered questionnaires and a wide range of biomedical examinations. Biomaterials were collected from all participants including serum, EDTA plasma, buffy coats, RNA and erythrocytes, urine, saliva, nasal swabs and stool. In 56,971 participants, an intensified examination programme was implemented. Whole-body 3T magnetic resonance imaging was performed in 30,861 participants on dedicated scanners. NAKO collects follow-up information on incident diseases through a combination of active follow-up using self-report via written questionnaires at 2–3 year intervals and passive follow-up via record linkages. All study participants are invited for re-examinations at the study centres in 4–5 year intervals. Thereby, longitudinal information on changes in risk factor profiles and in vascular, cardiac, metabolic, neurocognitive, pulmonary and sensory function is collected. NAKO is a major resource for population-based epidemiology to identify new and tailored strategies for early detection, prediction, prevention and treatment of major diseases for the next 30 years