Einfluss der Kryokonservierung auf die Immunantwort von Leukozyten

Die Messung von Zytokinen in Stimulationsexperimenten zur Bestimmung von Stärke und Umfang einer Immunreaktion werden in der Praxis häufig an kryokonserviertem Zellmaterial durchgeführt. Bisherige Untersuchungen zum Einfluss der Kryokonservierung auf die Zytokinexpression sind widersprüchlich. In den hier durchgeführten Experimenten wurden die Genexpression und/ oder Sekretion der Zytokine IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, GM-CSF, IFN-γ und TNF-α in frisch isolierten und kryokonservierten Immunzellen aus Blut (PBMC) eines gesunden Spenders bestimmt. Diese wurden mit den Immunsystem-aktivierenden Stoffen OKT-3 und Concanavalin A (Con A) stimuliert und für 4 bzw. 24 h kultiviert. Ein Vergleich der frisch-isolierten und kryokonservierten PBMC in Stimulationsexperimenten zeigt, (1) dass die Messung der Genexpression genauere Einblicke zu Beginn der einsetzenden Immunreaktion verschafft, als die Messung der ausgeschütteten Zytokine, (2) dass durch Einfrieren die Immunreaktion insbesondere zu diesem Zeitpunkt beeinflusst wird und (3) dass zu späteren Zeitpunkten die Konzentrationsbestimmung der Zytokine im Zellkulturüberstand das Mittel der Wahl ist.The measurement of cytokines in stimulation experiments with the aim to determine the strength and the complexitiy of an immune reaction is commonly carried out on cryopreserved cells. Previous studies, investigating the impact of cryopreservation on the cytokine expression, are inconsistent in their findings. Experiments conducted in this study determine the gene expression and/or secretion of IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, GM-CSF, IFN-γ and TNF-α in freshly isolated and cryopreserved PBMC of a healthy donor. The cells were stimulated with the substances OKT-3 and Concanavalin A (Con A) and cultured for 4h and 24h. The comparison of freshly isolated and cryopreserved cells in stimulation experiments showed (1) that the measurement of the gene expression offers a more accurate insight into the immune-reaction at early timepoints than the measurement of the secreted cytokines; (2) that freezing of the cells affect the immune response at this early stage and (3) that at later points the measurement of secreted proteins is the method of choice

Etablierung verschiedener Bead-basierter Multiplexmethoden mit einem Suspensions Array-System für molekulardiagnostische Zwecke

Die simultane Bestimmung mehrerer Analyten und die Erstellung komplexer Parameterprofi e erlangt immer größere Bedeutung in der heutigen Labordiagnostik. Die Bead-basierte Multiplexanalytik bietet hier eine flexible, schnelle und einfache Methode zur Erstellung individueller Analysen. Aufgrund der Vielzahl von Anwendungsmöglichkeiten, die diese moderne Nachweismethode im Bereich molekularbiologischer Fragestellungen bietet, ist die Etablierung der Beadbasierten Multiplexanalytik im Labor für Molekularbiologie und funktionelle Genomik der Technischen Hochschule Wildau von großem Nutzen. Zur Einarbeitung in das Testsystem wurden die Konzentrationen der Zytokine IL-2, IL-4, IFN-γ und TNF-α in Zellkulturüberständen mit kommerziellen Fertigsystemen gemessen und mit mRNA-Expressionsraten der gleichen Proben verglichen. Des Weiteren wurde ein Testsystem zum Nachweis von humanen Antikörpern der Klassen IgG und IgM sowie deren antigen-spezifischer Anteil in Zellkulturüberständen entwickelt. Außerdem konnte durch die erfolgreiche Detektion von DNA-gekoppelten Beads mittels markierter Oligonukleotidsequenz die Kopplung und die Anwendbarkeit der Methode auf Bindungsexperimente mit Nukleotidsequenzen gezeigt werdenThe simultaneous determination of multiple analytes and the generation of complex parameter profi les gains increasing importance in today’s laboratory diagnostics. The bead-based multiplex assay is offering a flexible, rapid and easy to handle method for the creation of individual analyses. Because of the multitude of applications this modern detection system offers, the establishment of the bead-based multiplex technique is of great benefit for the Laboratory for Molecular Biology and Functional Genomics of the Technical University of Applied Sciences Wildau. To familiarize with the testing system the concentrations of the cytokines IL-2, IL-4, IFN-γ and TNF-α in cell culture supernatant were analysed with commercially available assays and compared with mRNA expression ratios of the same samples. Furthermore a custom testing system was developed for the detection of human IgG and IgM antibodies and also antigen specific antibodies in cell culture supernatants. The assignability of the method to binding experiments with nucleotide sequences could be shown by the successful detection of DNA-modified beads by conjugated oligonucleotide

Transethnic Genome-Wide Association Study Provides Insights in the Genetic Architecture and Heritability of Long QT Syndrome

BACKGROUND: Long QT syndrome (LQTS) is a rare genetic disorder and a major preventable cause of sudden cardiac death in the young. A causal rare genetic variant with large effect size is identified in up to 80% of probands (genotype positive) and cascade family screening shows incomplete penetrance of genetic variants. Furthermore, a proportion of cases meeting diagnostic criteria for LQTS remain genetically elusive despite genetic testing of established genes (genotype negative). These observations raise the possibility that common genetic variants with small effect size contribute to the clinical picture of LQTS. This study aimed to characterize and quantify the contribution of common genetic variation to LQTS disease susceptibility. METHODS: We conducted genome-wide association studies followed by transethnic meta-analysis in 1656 unrelated patients with LQTS of European or Japanese ancestry and 9890 controls to identify susceptibility single nucleotide polymorphisms. We estimated the common variant heritability of LQTS and tested the genetic correlation between LQTS susceptibility and other cardiac traits. Furthermore, we tested the aggregate effect of the 68 single nucleotide polymorphisms previously associated with the QT-interval in the general population using a polygenic risk score. RESULTS: Genome-wide association analysis identified 3 loci associated with LQTS at genome-wide statistical significance (P&lt;5×10-8) near NOS1AP, KCNQ1, and KLF12, and 1 missense variant in KCNE1(p.Asp85Asn) at the suggestive threshold (P&lt;10-6). Heritability analyses showed that ≈15% of variance in overall LQTS susceptibility was attributable to common genetic variation (h2SNP 0.148; standard error 0.019). LQTS susceptibility showed a strong genome-wide genetic correlation with the QT-interval in the general population (rg=0.40; P=3.2×10-3). The polygenic risk score comprising common variants previously associated with the QT-interval in the general population was greater in LQTS cases compared with controls (P&lt;10-13), and it is notable that, among patients with LQTS, this polygenic risk score was greater in patients who were genotype negative compared with those who were genotype positive (P&lt;0.005). CONCLUSIONS: This work establishes an important role for common genetic variation in susceptibility to LQTS. We demonstrate overlap between genetic control of the QT-interval in the general population and genetic factors contributing to LQTS susceptibility. Using polygenic risk score analyses aggregating common genetic variants that modulate the QT-interval in the general population, we provide evidence for a polygenic architecture in genotype negative LQTS.</p

Enhancing rare variant interpretation in inherited arrhythmias through quantitative analysis of consortium disease cohorts and population controls.

PURPOSE: Stringent variant interpretation guidelines can lead to high rates of variants of uncertain significance (VUS) for genetically heterogeneous disease like long QT syndrome (LQTS) and Brugada syndrome (BrS). Quantitative and disease-specific customization of American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines can address this false negative rate. METHODS: We compared rare variant frequencies from 1847 LQTS (KCNQ1/KCNH2/SCN5A) and 3335 BrS (SCN5A) cases from the International LQTS/BrS Genetics Consortia to population-specific gnomAD data and developed disease-specific criteria for ACMG/AMP evidence classes-rarity (PM2/BS1 rules) and case enrichment of individual (PS4) and domain-specific (PM1) variants. RESULTS: Rare SCN5A variant prevalence differed between European (20.8%) and Japanese (8.9%) BrS patients (p = 5.7 × 10-18) and diagnosis with spontaneous (28.7%) versus induced (15.8%) Brugada type 1 electrocardiogram (ECG) (p = 1.3 × 10-13). Ion channel transmembrane regions and specific N-terminus (KCNH2) and C-terminus (KCNQ1/KCNH2) domains were characterized by high enrichment of case variants and >95% probability of pathogenicity. Applying the customized rules, 17.4% of European BrS and 74.8% of European LQTS cases had (likely) pathogenic variants, compared with estimated diagnostic yields (case excess over gnomAD) of 19.2%/82.1%, reducing VUS prevalence to close to background rare variant frequency. CONCLUSION: Large case-control data sets enable quantitative implementation of ACMG/AMP guidelines and increased sensitivity for inherited arrhythmia genetic testing

In Vitro Evaluation of Glycoengineered RSV-F in the Human Artificial Lymph Node Reactor

Subunit vaccines often require adjuvants to elicit sustained immune activity. Here, a method is described to evaluate the efficacy of single vaccine candidates in the preclinical stage based on cytokine and gene expression analysis. As a model, the recombinant human respiratory syncytial virus (RSV) fusion protein (RSV-F) was produced in CHO cells. For comparison, wild-type and glycoengineered, afucosylated RSV-F were established. Both glycoprotein vaccines were tested in a commercial Human Artificial Lymph Node in vitro model (HuALN®). The analysis of six key cytokines in cell culture supernatants showed well-balanced immune responses for the afucosylated RSV-F, while immune response of wild-type RSV-F was more Th1 accentuated. In particular, stronger and specific secretion of interleukin-4 after each round of re-stimulation underlined higher potency and efficacy of the afucosylated vaccine candidate. Comprehensive gene expression analysis by nCounter gene expression assay confirmed the stronger onset of the immunologic reaction in stimulation experiments with the afucosylated vaccine in comparison to wild-type RSV-F and particularly revealed prominent activation of Th17 related genes, innate immunity, and comprehensive activation of humoral immunity. We, therefore, show that our method is suited to distinguish the potency of two vaccine candidates with minor structural differences

Transethnic Genome-Wide Association Study Provides Insights in the Genetic Architecture and Heritability of Long QT Syndrome

Supplemental Digital Content is available in the text. We conducted genome-wide association studies followed by transethnic meta-analysis in 1656 unrelated patients with LQTS of European or Japanese ancestry and 9890 controls to identify susceptibility single nucleotide polymorphisms. We estimated the common variant heritability of LQTS and tested the genetic correlation between LQTS susceptibility and other cardiac traits. Furthermore, we tested the aggregate effect of the 68 single nucleotide polymorphisms previously associated with the QT-interval in the general population using a polygenic risk score. Genome-wide association analysis identified 3 loci associated with LQTS at genome-wide statistical significance (P <5×10 −8) near NOS1AP, KCNQ1, and KLF12, and 1 missense variant in KCNE1 (p.Asp85Asn) at the suggestive threshold (P <10 −6). Heritability analyses showed that ≈15% of variance in overall LQTS susceptibility was attributable to common genetic variation (h2SNP 0.148; standard error 0.019). LQTS susceptibility showed a strong genome-wide genetic correlation with the QT-interval in the general population (r=0.40; P =3.2×10 −3). The polygenic risk score comprising common variants previously associated with the QT-interval in the general population was greater in LQTS cases compared with controls (P <10−13), and it is notable that, among patients with LQTS, this polygenic risk score was greater in patients who were genotype negative compared with those who were genotype positive (P <0.005). This work establishes an important role for common genetic variation in susceptibility to LQTS. We demonstrate overlap between genetic control of the QT-interval in the general population and genetic factors contributing to LQTS susceptibility. Using polygenic risk score analyses aggregating common genetic variants that modulate the QT-interval in the general population, we provide evidence for a polygenic architecture in genotype negative LQTS

Chromosome Xq23 is associated with lower atherogenic lipid concentrations and favorable cardiometabolic indices

Abstract Autosomal genetic analyses of blood lipids have yielded key insights for coronary heart disease (CHD). However, X chromosome genetic variation is understudied for blood lipids in large sample sizes. We now analyze genetic and blood lipid data in a high-coverage whole X chromosome sequencing study of 65,322 multi-ancestry participants and perform replication among 456,893 European participants. Common alleles on chromosome Xq23 are strongly associated with reduced total cholesterol, LDL cholesterol, and triglycerides (min P = 8.5 × 10−72), with similar effects for males and females. Chromosome Xq23 lipid-lowering alleles are associated with reduced odds for CHD among 42,545 cases and 591,247 controls (P = 1.7 × 10−4), and reduced odds for diabetes mellitus type 2 among 54,095 cases and 573,885 controls (P = 1.4 × 10−5). Although we observe an association with increased BMI, waist-to-hip ratio adjusted for BMI is reduced, bioimpedance analyses indicate increased gluteofemoral fat, and abdominal MRI analyses indicate reduced visceral adiposity. Co-localization analyses strongly correlate increased CHRDL1 gene expression, particularly in adipose tissue, with reduced concentrations of blood lipids