Genome-wide association study of panic disorder reveals genetic overlap with neuroticism and depression.

Panic disorder (PD) has a lifetime prevalence of 2-4% and heritability estimates of 40%. The contributory genetic variants remain largely unknown, with few and inconsistent loci having been reported. The present report describes the largest genome-wide association study (GWAS) of PD to date comprising genome-wide genotype data of 2248 clinically well-characterized PD patients and 7992 ethnically matched controls. The samples originated from four European countries (Denmark, Estonia, Germany, and Sweden). Standard GWAS quality control procedures were conducted on each individual dataset, and imputation was performed using the 1000 Genomes Project reference panel. A meta-analysis was then performed using the Ricopili pipeline. No genome-wide significant locus was identified. Leave-one-out analyses generated highly significant polygenic risk scores (PRS) (explained variance of up to 2.6%). Linkage disequilibrium (LD) score regression analysis of the GWAS data showed that the estimated heritability for PD was 28.0-34.2%. After correction for multiple testing, a significant genetic correlation was found between PD and major depressive disorder, depressive symptoms, and neuroticism. A total of 255 single-nucleotide polymorphisms (SNPs) with p < 1 × 10-4 were followed up in an independent sample of 2408 PD patients and 228,470 controls from Denmark, Iceland and the Netherlands. In the combined analysis, SNP rs144783209 showed the strongest association with PD (pcomb = 3.10 × 10-7). Sign tests revealed a significant enrichment of SNPs with a discovery p-value of <0.0001 in the combined follow up cohort (p = 0.048). The present integrative analysis represents a major step towards the elucidation of the genetic susceptibility to PD.

Chronic filarial infection provides protection against bacterial sepsis by functionally reprogramming macrophages.

Helminths immunomodulate their hosts and induce a regulatory, anti-inflammatory milieu that prevents allergies and autoimmune diseases. Helminth immunomodulation may benefit sepsis outcome by preventing exacerbated inflammation and severe pathology, but the influence on bacterial clearance remains unclear. To address this, mice were chronically infected with the filarial nematode Litomosoides sigmodontis (L.s.) and the outcome of acute systemic inflammation caused by i.p. Escherichia coli injection was determined. L.s. infection significantly improved E. coli-induced hypothermia, bacterial clearance and sepsis survival and correlated with reduced concentrations of associated pro-inflammatory cytokines/chemokines and a less pronounced pro-inflammatory macrophage gene expression profile. Improved sepsis outcome in L.s.-infected animals was mediated by macrophages, but independent of the alternatively activated macrophage subset. Endosymbiotic Wolbachia bacteria that are present in most human pathogenic filariae, as well as L.s., signal via TLR2 and modulate macrophage function. Here, gene expression profiles of peritoneal macrophages from L.s.-infected mice revealed a downregulation of genes involved in TLR signaling, and pulsing of macrophages in vitro with L.s. extract reduced LPS-triggered activation. Subsequent transfer improved sepsis outcome in naïve mice in a Wolbachia- and TLR2-dependent manner. In vivo, phagocytosis was increased in macrophages from L.s.-infected wild type, but not TLR2-deficient animals. In association, L.s. infection neither improved bacterial clearance in TLR2-deficient animals nor ameliorated E. coli-induced hypothermia and sepsis survival. These results indicate that chronic L.s. infection has a dual beneficial effect on bacterial sepsis, reducing pro-inflammatory immune responses and improving bacterial control. Thus, helminths and their antigens may not only improve the outcome of autoimmune and allergic diseases, but may also present new therapeutic approaches for acute inflammatory diseases that do not impair bacterial control.

Escherichia coli-induced immune paralysis is not exacerbated during chronic filarial infection.

Sepsis initially starts with a systemic inflammatory response (SIRS phase) and is followed by a compensatory anti-inflammatory response syndrome (CARS) that causes impaired adaptive T-cell immunity, immune paralysis and an increased susceptibility to secondary infections. In contrast, parasitic filariae release thousands of microfilariae into the peripheral blood without triggering inflammation, as they induce regulatory, anti-inflammatory host responses. Hence, we investigated the impact of chronic filarial infection on adaptive T-cell responses during the SIRS and CARS phases of a systemic bacterial infection and analysed the development of T-cell paralysis following a subsequent adenovirus challenge in BALB/c mice. Chronic filarial infection impaired adenovirus-specific CD8(+) T-cell cytotoxicity and interferon-γ responses in the absence of a bacterial challenge and led to higher numbers of splenic CTLA-4(+)  CD4(+) T cells, whereas splenic T-cell expression of CD69 and CD62 ligand, serum cytokine levels and regulatory T-cell frequencies were comparable to naive controls. Irrespective of filarial infection, the SIRS phase dominated 6-24 hr after intravenous Escherichia coli challenge with increased T-cell activation and pro-inflammatory cytokine production, whereas the CARS phase occurred 6 days post E. coli challenge and correlated with high levels of transforming growth factor-ß and increased CD62 ligand T-cell expression. Escherichia coli-induced impairment of adenovirus-specific CD8(+) T-cell cytotoxicity and interferon-γ production was not additionally impaired by chronic filarial infection. This suggests that filarial immunoregulation does not exacerbate E. coli-induced T-cell paralysis.

Partitioning behavior of short DNA fragments in polymer/salt aqueous two-phase systems.

The development of liquid biopsy as a minimally invasive technique for tumor profiling has created a need for efficient biomarker extraction systems from body fluids. The analysis of circulating cell-free DNA (cfDNA) is especially promising, but the low amounts and high fragmentation of cfDNA found in plasma pose challenges to its isolation. While the potential of aqueous two-phase systems (ATPS) for the extraction and purification of various biomolecules has already been successfully established, there is limited literature on the applicability of these findings to short cfDNA-like fragments. This study presents the partitioning behavior of a 160 bp DNA fragment in polyethylene glycol (PEG)/salt ATPS at pH 7.4. The effect of PEG molecular weight, tie-line length, neutral salt additives, and phase volume ratio is evaluated to maximize DNA recovery. Selected ATPS containing a synthetic plasma solution spiked with human serum albumin and immunoglobulin G are tested to determine the separation of DNA fragments from the main plasma protein fraction. By adding 1.5% (w/w) NaCl to a 17.7% (w/w) PEG 400/17.3% (w/w) phosphate ATPS, 88% DNA recovery was achieved in the salt-rich bottom phase while over 99% of the protein was removed.

Pathogen-specific innate immune response patterns are distinctly affected by genetic diversity.

Innate immune responses vary by pathogen and host genetics. We analyze quantitative trait loci (eQTLs) and transcriptomes of monocytes from 215 individuals stimulated by fungal, Gram-negative or Gram-positive bacterial pathogens. We identify conserved monocyte responses to bacterial pathogens and a distinct antifungal response. These include 745 response eQTLs (reQTLs) and corresponding genes with pathogen-specific effects, which we find first in samples of male donors and subsequently confirm for selected reQTLs in females. reQTLs affect predominantly upregulated genes that regulate immune response via e.g., NOD-like, C-type lectin, Toll-like and complement receptor-signaling pathways. Hence, reQTLs provide a functional explanation for individual differences in innate response patterns. Our identified reQTLs are also associated with cancer, autoimmunity, inflammatory and infectious diseases as shown by external genome-wide association studies. Thus, reQTLs help to explain interindividual variation in immune response to infection and provide candidate genes for variants associated with a range of diseases.

A genome-wide association study with tissue transcriptomics identifies genetic drivers for classic bladder exstrophy.

Classic bladder exstrophy represents the most severe end of all human congenital anomalies of the kidney and urinary tract and is associated with bladder cancer susceptibility. Previous genetic studies identified one locus to be involved in classic bladder exstrophy, but were limited to a restrict number of cohort. Here we show the largest classic bladder exstrophy genome-wide association analysis to date where we identify eight genome-wide significant loci, seven of which are novel. In these regions reside ten coding and four non-coding genes. Among the coding genes is EFNA1, strongly expressed in mouse embryonic genital tubercle, urethra, and primitive bladder. Re-sequence of EFNA1 in the investigated classic bladder exstrophy cohort of our study displays an enrichment of rare protein altering variants. We show that all coding genes are expressed and/or significantly regulated in both mouse and human embryonic developmental bladder stages. Furthermore, nine of the coding genes residing in the regions of genome-wide significance are differentially expressed in bladder cancers. Our data suggest genetic drivers for classic bladder exstrophy, as well as a possible role for these drivers to relevant bladder cancer susceptibility.

TGFß depletion does neither modulate acute E. coli-induced inflammatory immune responses nor impair the protective effect by chronic filarial infection.

TGFß is an anti-inflammatory molecule that suppresses pro-inflammatory immune responses. Previously, we demonstrated that chronic filarial infection has a beneficial impact on Escherichia coli-induced sepsis. In the present study, we investigated whether this protective effect is dependent on TGFß signaling and whether depletion of TGFß before E. coli challenge alters the early course of sepsis per se. In vivo depletion of TGFß before E. coli challenge did not alter levels of pro-inflammatory cytokines/chemokines and did neither increase the bacterial burden nor worsen E. coli-induced hypothermia six hours post E. coli challenge. Similarly, in the co-infection model, despite TGFß depletion, mice infected with the filarial nematode Litomosoides sigmodontis exhibited milder E. coli-induced hypothermia, reduced bacterial load and pro-inflammatory immune responses. Thus, we conclude that TGFß is not essentially modulating the initial pro-inflammatory phase during sepsis and that the protective effect of a chronic filarial infection against sepsis is independent of TGFß signaling.

Susceptibility to L. sigmodontis infection is highest in animals lacking IL-4R/IL-5 compared to single knockouts of IL-4R, IL-5 or eosinophils.

BACKGROUND: Mice are susceptible to infections with the rodent filarial nematode Litomosoides sigmodontis and develop immune responses that resemble those of human filarial infections. Thus, the L. sigmodontis model is used to study filarial immunomodulation, protective immune responses against filariae and to screen drug candidates for human filarial diseases. While previous studies showed that type 2 immune responses are protective against L. sigmodontis, the present study directly compared the impact of eosinophils, IL-5, and the IL-4R on the outcome of L. sigmodontis infection. METHODS: Susceptible wildtype (WT) BALB/c mice, BALB/c mice lacking eosinophils (dblGATA mice), IL-5-/- mice, IL-4R-/- mice and IL-4R-/-/IL-5-/- mice were infected with L. sigmodontis. Analyses were performed during the peak of microfilaremia in WT animals (71 dpi) as well as after IL-4R-/-/IL-5-/- mice showed a decline in microfilaremia (119 dpi) and included adult worm counts, peripheral blood microfilariae levels, cytokine production from thoracic cavity lavage, the site of adult worm residence, and quantification of major immune cell types within the thoracic cavity and spleen. RESULTS: Our study reveals that thoracic cavity eosinophil numbers correlated negatively with the adult worm burden, whereas correlations of alternatively activated macrophage (AAM) numbers with the adult worm burden (positive correlation) were likely attributed to the accompanied changes in eosinophil numbers. IL-4R-/-/IL-5-/- mice exhibited an enhanced embryogenesis achieving the highest microfilaremia with all animals becoming microfilariae positive and had an increased adult worm burden combined with a prolonged adult worm survival. CONCLUSIONS: These data indicate that mice deficient for IL-4R-/-/IL-5-/- have the highest susceptibility for L. sigmodontis infection, which resulted in an earlier onset of microfilaremia, development of microfilaremia in all animals with highest microfilariae loads, and an extended adult worm survival.

Genome-wide transcriptome induced by nickel in human monocytes.

UNLABELLED: Nickel-containing alloys are frequently used in the biomedical field, although, owing to corrosive processes metal ion leaching is inevitable. Due to nickel ion (Ni(2+)) leaching several adverse effects are described in the literature. However, only a few studies evaluated the genetic profile of Ni(2+) in human cells which is of great importance since nickel-induced effects differ between humans and mice as a result of species-specific receptor variability. Thus, we investigated gene expression induced by Ni(2+)in human monocytes using a transcriptome-wide approach determining new target genes implicated in nickel-induced pathologies. Monocytes were isolated from healthy volunteers of Central European origin using stringent inclusion criteria. Cells were challenged with different Ni(2+) concentrations. Array-based gene expression analysis was performed comprising more than 47,000 transcripts followed by pathway analyses. Transcriptional data were validated by protein and cell surface markers. Ni(2+) significantly influenced the expression of 1385 transcripts in a dose-dependent manner. Apart from known targets (CCL20↑, PTGS2↑, MTs↑, SLCs↑), we identified new candidates implicated in Ni(2+)-elicited processes (various microRNAs↑, INSIG1↑, NAMPT↑, MS4A6A↓, DHRS9↓). Several of these transcripts correspond to immunity, inflammation and were shown to be involved in cellular reactions related to hypersensitivity, cancer, colitis, and encephalitis. Moreover, 459 canonical pathways/signaling, 500 pathologies and 2687 upstream regulators were detected. Protein results validated our findings. To our knowledge, the present systematic transcriptome-wide expression study is the first which explored Ni(2+)-elicited cell responses in human primary monocytes identifying new target genes, pathways and upstream regulators of relevance to diagnostic and therapeutic strategies. STATEMENT OF SIGNIFICANCE: Nickel is widely applied in the biomedical field, although several adverse effects are documented in the literature due to nickel ion (Ni(2+)) leaching. In humans, allergic reactions like contact dermatitis are the most common adverse effect to Ni(2+), whereas serious concerns relate to possible systemic and carcinogenic activities. Using a systematic genome-wide transcriptional approach in human primary monocytes unveil new target genes, pathways and upstream regulators implicated in nickel-elicited immune response which are of significance to diagnostic and therapeutic strategies. This approach provides new information of how host-derived immune response contributes to the interaction with antigens and supports the interplay between metal ions and systemic diseases.

Genome-wide transcriptome induced by Porphyromonas gingivalis LPS supports the notion of host-derived periodontal destruction and its association with systemic diseases.

Chronic periodontitis (CP) is a prevalent pathogen-associated inflammatory disorder characterized by the destruction of tooth-supporting tissues, and linked to several systemic diseases. Both the periodontopathogen Porphyromonas gingivalis (Pg), and the genetically determined host immune response, are hypothesized to play a crucial role in this association. To identify new target genes for CP and its associated systemic diseases, we investigated the transcriptome induced by Pg in human monocytes using a genome-wide approach. Monocytes were isolated from healthy male volunteers of European origin and challenged with the Pg virulence factor LPS. Array-based gene expression analysis comprising >47,000 transcripts was performed followed by pathway analyses. Transcriptional data were validated by protein and cell surface markers. LPS Pg challenge led to the significant induction of 902 transcripts. Besides known periodontitis-associated targets, several new candidates were identified (CCL23↑, INDO↑, GBP 1/4↑, CFB↑, ISG20↑, MIR155HG↑, DHRS9↓). Moreover, various transcripts correspond to the host immune response, and have been linked to cancer, atherosclerosis and arthritis, thus highlighting the systemic impact of CP. Protein data of immunological markers validated our results. The present findings expand understanding of Pg elicited immune responses, and indicate new target genes and pathways of relevance to diagnostic and therapeutic strategies.