Study protocol of the HessenKohorte2042: a prospective, longitudinal cohort study characterising quality of life in people with Parkinson's disease and their caregivers using a bio-psycho-social approach.

INTRODUCTION: Quality of life (QoL) is of paramount importance as an outcome to monitor and guide therapies for people with Parkinson's disease (PwPD). In particular, due to the heterogeneous symptoms that PwPD may experience during their disease course, QoL can deteriorate not only in patients but also in their caregivers, with a variety of psychosocial consequences. However, there is a lack of longitudinal studies that explore how QoL evolves over time and what factors are significant. Furthermore, holistic approaches that consider bio-psycho-social determinants are rare. In the worst cases, these gaps can lead to suboptimal care and therefore unmet needs for patients and their caregivers, resulting in unnecessary symptom burden and increased healthcare costs for society. METHODS AND ANALYSIS: This prospective, longitudinal study will follow 1000 PwPD along with their caregivers for 20 years, with up to 40 semi-annual assessments. Patient data and sample collection will include clinical assessments, self-reported outcome measures focusing on QoL, biospecimen collection and MRI. Caregiver burden will be systematically assessed through self-administered questionnaires. The use of digitised surveys will allow efficient data collection and convenient assessment at home. Our primary objective is to attain a holistic understanding of QoL in PwPD and establish a tool to measure it. The secondary objective is to explore the psycho-social and biological variables associated with QoL of patients and caregivers over the progression of the disease. This will provide key information for diagnostic and prognostic prediction, therapeutic patient stratification and adaptation of therapy in the future. ETHICS AND DISSEMINATION: The study was approved by the local ethics committee of the University Hospital of Marburg (study number: 209/19). The results will be disseminated by means of publication in peer-reviewed journals, international conference contributions, annual patient meetings and a dedicated website. TRIAL REGISTRATION NUMBER: German Clinical Trials Register (DRKS00023598).

Identification of extracellular vesicle microRNA signatures specifically linked to inflammatory and metabolic mechanisms in obesity-associated low type-2 asthma.

RATIONALE AND OBJECTIVE: Plasma extracellular vesicles (EVs) represent a vital source of molecular information about health and disease states. Due to their heterogenous cellular sources, EVs and their cargo may predict specific pathomechanisms behind disease phenotypes. Here we aimed to utilize EV microRNA (miRNA) signatures to gain new insights into underlying molecular mechanisms of obesity-associated low type-2 asthma. METHODS: Obese low type-2 asthma (OA) and non-obese low type-2 asthma (NOA) patients were selected from an asthma cohort conjointly with healthy controls. Plasma EVs were isolated and characterised by nanoparticle tracking analysis. EV-associated small RNAs were extracted, sequenced and bioinformatically analysed. RESULTS: Based on EV miRNA expression profiles, a clear distinction between the three study groups could be established using a principal component analysis. Integrative pathway analysis of potential target genes of the differentially expressed miRNAs revealed inflammatory cytokines (e.g., interleukin-6, transforming growth factor-beta, interferons) and metabolic factors (e.g., insulin, leptin) signalling pathways to be specifically associated with OA. The miR-17-92 and miR-106a-363 clusters were significantly enriched only in OA. These miRNA clusters exhibited discrete bivariate correlations with several key laboratory (e.g., C-reactive protein) and lung function parameters. Plasma EV miRNA signatures mirrored blood-derived CD4+ T-cell transcriptome data, but achieved an even higher sensitivity in identifying specifically affected biological pathways. CONCLUSION: The identified plasma EV miRNA signatures and particularly the miR-17-92 and -106a-363 clusters were capable to disentangle specific mechanisms of the obesity-associated low type-2 asthma phenotype, which may serve as basis for stratified treatment development.

IL-17A-producing CD8+ T cells promote PDAC via induction of inflammatory cancer-associated fibroblasts.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant desmoplastic stroma composed of cancer-associated fibroblasts (CAF) and interspersed immune cells. A non-canonical CD8+ T-cell subpopulation producing IL-17A (Tc17) promotes autoimmunity and has been identified in tumours. Here, we evaluated the Tc17 role in PDAC. DESIGN: Infiltration of Tc17 cells in PDAC tissue was correlated with patient overall survival and tumour stage. Wild-type (WT) or Il17ra-/- quiescent pancreatic stellate cells (qPSC) were exposed to conditional media obtained from Tc17 cells (Tc17-CM); moreover, co-culture of Tc17-CM-induced inflammatory (i)CAF (Tc17-iCAF) with tumour cells was performed. IL-17A/F-, IL-17RA-, RAG1-deficient and Foxn1nu/nu mice were used to study the Tc17 role in subcutaneous and orthotopic PDAC mouse models. RESULTS: Increased abundance of Tc17 cells highly correlated with reduced survival and advanced tumour stage in PDAC. Tc17-CM induced iCAF differentiation as assessed by the expression of iCAF-associated genes via synergism of IL-17A and TNF. Accordingly, IL-17RA controlled the responsiveness of qPSC to Tc17-CM. Pancreatic tumour cells co-cultured with Tc17-iCAF displayed enhanced proliferation and increased expression of genes implicated in proliferation, metabolism and protection from apoptosis. Tc17-iCAF accelerated growth of mouse and human tumours in Rag1-/- and Foxn1nu/nu mice, respectively. Finally, Il17ra-expressed by fibroblasts was required for Tc17-driven tumour growth in vivo. CONCLUSIONS: We identified Tc17 as a novel protumourigenic CD8+ T-cell subtype in PDAC, which accelerated tumour growth via IL-17RA-dependent stroma modification. We described a crosstalk between three cell types, Tc17, fibroblasts and tumour cells, promoting PDAC progression, which resulted in poor prognosis for patients.

Pericentromeric satellite lncRNAs are induced in cancer-associated fibroblasts and regulate their functions in lung tumorigenesis.

The abnormal tumor microenvironment (TME) often dictates the therapeutic response of cancer to chemo- and immuno-therapy. Aberrant expression of pericentromeric satellite repeats has been reported for epithelial cancers, including lung cancer. However, the transcription of tandemly repetitive elements in stromal cells of the TME has been unappreciated, limiting the optimal use of satellite transcripts as biomarkers or anti-cancer targets. We found that transcription of pericentromeric satellite DNA (satDNA) in mouse and human lung adenocarcinoma was observed in cancer-associated fibroblasts (CAFs). In vivo, lung fibroblasts expressed pericentromeric satellite repeats HS2/HS3 specifically in tumors. In vitro, transcription of satDNA was induced in lung fibroblasts in response to TGFß, IL1α, matrix stiffness, direct contact with tumor cells and treatment with chemotherapeutic drugs. Single-cell transcriptome analysis of human lung adenocarcinoma confirmed that CAFs were the cell type with the highest number of satellite transcripts. Human HS2/HS3 pericentromeric transcripts were detected in the nucleus, cytoplasm, extracellularly and co-localized with extracellular vesicles in situ in human biopsies and activated fibroblasts in vitro. The transcripts were transmitted into recipient cells and entered their nuclei. Knock-down of satellite transcripts in human lung fibroblasts attenuated cellular senescence and blocked the formation of an inflammatory CAFs phenotype which resulted in the inhibition of their pro-tumorigenic functions. In sum, our data suggest that satellite long non-coding (lnc) RNAs are induced in CAFs, regulate expression of inflammatory genes and can be secreted from the cells, which potentially might present a new element of cell-cell communication in the TME.

Immune Responsiveness to LPS Determines Risk of Childhood Wheeze and Asthma in 17q21 Risk Allele Carriers.

Rationale: In murine models, microbial exposures induce protection from experimental allergic asthma through innate immunity. Objectives: Our aim was to assess the association of early life innate immunity with the development of asthma in children at risk. Methods: In the PASTURE farm birth cohort, innate T-helper cell type 2 (Th2), Th1, and Th17 cytokine expression at age 1 year was measured after stimulation of peripheral blood mononuclear cells with LPS in n = 445 children. Children at risk of asthma were defined based on single-nucleotide polymorphisms at the 17q21 asthma gene locus. Specifically, we used the SNP rs7216389 in the GSDMB gene. Wheeze in the first year of life was assessed by weekly diaries and asthma by questionnaire at age 6 years. Measurements and Main Results: Not all cytokines were detectable in all children after LPS stimulation. When classifying detectability of cytokines by latent class analysis, carrying the 17q21 risk allele rs7216389 was associated with risk of wheeze only in the class with the lowest level of LPS-induced activation: odds ratio (OR), 1.89; 95% confidence interval [CI], 1.13-3.16; P = 0.015. In contrast, in children with high cytokine activation after LPS stimulation, no association of the 17q21 risk allele with wheeze (OR, 0.63; 95% CI, 0.29-1.40; P = 0.258, P = 0.034 for interaction) or school-age asthma was observed. In these children, consumption of unprocessed cow's milk was associated with higher cytokine activation (OR, 3.37; 95% CI, 1.56-7.30; P = 0.002), which was in part mediated by the gut microbiome. Conclusions: These findings suggest that within the 17q21 genotype, asthma risk can be mitigated by activated immune responses after innate stimulation, which is partly mediated by a gut-immune axis.

Differential Regulation of Interferon Signaling Pathways in CD4+ T Cells of the Low Type-2 Obesity-Associated Asthma Phenotype.

In the era of personalized medicine, insights into the molecular mechanisms that differentially contribute to disease phenotypes, such as asthma phenotypes including obesity-associated asthma, are urgently needed. Peripheral blood was drawn from 10 obese, non-atopic asthmatic adults with a high body mass index (BMI; 36.67 ± 6.90); 10 non-obese, non-atopic asthmatic adults with normal BMI (23.88 ± 2.73); and 10 healthy controls with normal BMI (23.62 ± 3.74). All asthmatic patients were considered to represent a low type-2 asthma phenotype according to selective clinical parameters. RNA sequencing (RNA-Seq) was conducted on peripheral blood CD4+ T cells. Thousands of differentially expressed genes were identified in both asthma groups compared with heathy controls. The expression of interferon (IFN)-stimulated genes associated with IFN-related signaling pathways was specifically affected in obese asthmatics, while the gap junction and G protein-coupled receptor (GPCR) ligand binding pathways were enriched in both asthma groups. Furthermore, obesity gene markers were also upregulated in CD4+ T cells from obese asthmatics compared with the two other groups. Additionally, the enriched genes of the three abovementioned pathways showed a unique correlation pattern with various laboratory and clinical parameters. The specific activation of IFN-related signaling and viral infection pathways might provide a novel view of the molecular mechanisms associated with the development of the low type-2 obesity-associated asthma phenotype, which is a step ahead in the development of new stratified therapeutic approaches.

Matrix stiffness drives stromal autophagy and promotes formation of a protumorigenic niche.

Increased stiffness of solid tissues has long been recognized as a diagnostic feature of several pathologies, most notably malignant diseases. In fact, it is now well established that elevated tissue rigidity enhances disease progression and aggressiveness and is associated with a poor prognosis in patients as documented, for instance, for lung fibrosis or the highly desmoplastic cancer of the pancreas. The underlying mechanisms of the interplay between physical properties and cellular behavior are, however, not very well understood. Here, we have found that switching culture conditions from soft to stiff substrates is sufficient to evoke (macro) autophagy in various fibroblast types. Mechanistically, this is brought about by stiffness-sensing through an Integrin αV-focal adhesion kinase module resulting in sequestration and posttranslational stabilization of the metabolic master regulator AMPKα at focal adhesions, leading to the subsequent induction of autophagy. Importantly, stiffness-induced autophagy in stromal cells such as fibroblasts and stellate cells critically supports growth of adjacent cancer cells in vitro and in vivo. This process is Integrin αV dependent, opening possibilities for targeting tumor-stroma crosstalk. Our data thus reveal that the mere change in mechanical tissue properties is sufficient to metabolically reprogram stromal cell populations, generating a tumor-supportive metabolic niche.

DYRK3 contributes to differentiation and hypoxic control in neuroblastoma.

Neuroblastoma (NB), a pediatric cancer of the peripheral sympathetic nervous system, represents the most frequent solid malignancy in infants. Treatment of high-risk patients is still challenging and, depending on the genetic make-up and involved risk factors, the 5-year survival rate can drop to only 30%. Here, we found that the expression of the Dual Specificity Tyrosine Phosphorylation Regulated Kinase 3 (DYRK3) is increased in NB and is associated with decreased survival in NB patients. We further identified DYRK3 as a cytoplasmic kinase in NB cells and found that its levels are increased by hypoxic conditions. Further mechanistic studies revealed that DYRK3 acts as a negative regulator of HIF-driven transcriptional responses, suggesting that it functions in a negative feedback loop controlling the hypoxic response. Moreover, DYRK3 negatively impacted on NB cell differentiation, proposing an oncogenic role of this kinase in the etiology of NB. In summary, we describe novel functions of the DYRK3 kinase in NB, which will help to further improve the understanding of this disease eventually leading to the design of improved therapeutic concepts.

The Hygiene Hypothesis and New Perspectives-Current Challenges Meeting an Old Postulate.

During its 30 years history, the Hygiene Hypothesis has shown itself to be adaptable whenever it has been challenged by new scientific developments and this is a still a continuously ongoing process. In this regard, the mini review aims to discuss some selected new developments in relation to their impact on further fine-tuning and expansion of the Hygiene Hypothesis. This will include the role of recently discovered classes of innate and adaptive immune cells that challenges the old Th1/Th2 paradigm, the applicability of the Hygiene Hypothesis to newly identified allergy/asthma phenotypes with diverse underlying pathomechanistic endotypes, and the increasing knowledge derived from epigenetic studies that leads to better understanding of mechanisms involved in the translation of environmental impacts on biological systems. Further, we discuss in brief the expansion of the Hygiene Hypothesis to other disease areas like psychiatric disorders and cancer and conclude that the continuously developing Hygiene Hypothesis may provide a more generalized explanation for health burden in highly industrialized countries also relation to global changes.

The Hygiene Hypothesis - Learning From but Not Living in the Past.

Postulated by Strachan more than 30 years ago, the Hygiene Hypothesis has undergone many revisions and adaptations. This review journeys back to the beginnings of the Hygiene Hypothesis and describes the most important landmarks in its development considering the many aspects that have refined and generalized the Hygiene Hypothesis over time. From an epidemiological perspective, the Hygiene Hypothesis advanced to a comprehensive concept expanding beyond the initial focus on allergies. The Hygiene Hypothesis comprise immunological, microbiological and evolutionary aspects. Thus, the original postulate developed into a holistic model that explains the impact of post-modern life-style on humans, who initially evolved in close proximity to a more natural environment. Focusing on diet and the microbiome as the most prominent exogenous influences we describe these discrepancies and the resulting health outcomes and point to potential solutions to reestablish the immunological homeostasis that frequently have been lost in people living in developed societies.

Constitutive immune activity promotes JNK- and FoxO-dependent remodeling of Drosophila airways.

Extensive remodeling of the airways is a major characteristic of chronic inflammatory lung diseases such as asthma or chronic obstructive pulmonary disease (COPD). To elucidate the importance of a deregulated immune response in the airways for remodeling processes, we established a matching Drosophila model. Here, triggering the Imd (immune deficiency) pathway in tracheal cells induced organ-wide remodeling. This structural remodeling comprises disorganization of epithelial structures and comprehensive epithelial thickening. We show that these structural changes do not depend on the Imd pathway's canonical branch terminating on nuclear factor κB (NF-κB) activation. Instead, activation of a different segment of the Imd pathway that branches off downstream of Tak1 and comprises activation of c-Jun N-terminal kinase (JNK) and forkhead transcription factor of the O subgroup (FoxO) signaling is necessary and sufficient to mediate the observed structural changes of the airways. Our findings imply that targeting JNK and FoxO signaling in the airways could be a promising strategy to interfere with disease-associated airway remodeling processes.

Author Correction: Farm-like indoor microbiota in non-farm homes protects children from asthma development.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Farm-like indoor microbiota in non-farm homes protects children from asthma development.

Asthma prevalence has increased in epidemic proportions with urbanization, but growing up on traditional farms offers protection even today1. The asthma-protective effect of farms appears to be associated with rich home dust microbiota2,3, which could be used to model a health-promoting indoor microbiome. Here we show by modeling differences in house dust microbiota composition between farm and non-farm homes of Finnish birth cohorts4 that in children who grow up in non-farm homes, asthma risk decreases as the similarity of their home bacterial microbiota composition to that of farm homes increases. The protective microbiota had a low abundance of Streptococcaceae relative to outdoor-associated bacterial taxa. The protective effect was independent of richness and total bacterial load and was associated with reduced proinflammatory cytokine responses against bacterial cell wall components ex vivo. We were able to reproduce these findings in a study among rural German children2 and showed that children living in German non-farm homes with an indoor microbiota more similar to Finnish farm homes have decreased asthma risk. The indoor dust microbiota composition appears to be a definable, reproducible predictor of asthma risk and a potential modifiable target for asthma prevention.

Progranulin serum levels and gene expression in subcutaneous vs visceral adipose tissue of severely obese patients undergoing bariatric surgery.

BACKGROUND: Progranulin represents an adipokine putatively mediating insulin resistance and inflammation. Data in humans are sparse, and the source of circulating progranulin in obesity is unknown. OBJECTIVES: Serum progranulin concentrations and subcutaneous (sc) as well as visceral (vis) adipose tissue (AT) progranulin expression were quantified in a large cohort of patients with obesity undergoing bariatric surgery (BS) (n = 153) or a low-calorie diet (LCD) (n = 121). COHORTS AND METHODS: Paired serum and AT mRNA samples were obtained from patients with severe obesity undergoing BS (ROBS cohort; Research in Obesity and Bariatric Surgery). Serum progranulin was measured by ELISA in both cohorts, and AT mRNA expression was analysed by quantitative real-time PCR in bariatric patients. RESULTS: There was no gender-specific effect in serum progranulin or AT progranulin expression. Importantly, circulating progranulin was independent from adipose tissue gene expression in paired samples. sc AT progranulin expression was higher than in vis AT (P = 0.027), and there was a positive correlation between sc AT and vis AT gene expression (P < 0.001; r = +0.34). Serum progranulin strongly and rapidly increased after BS within 3 days and remained elevated up to 12 months. Serum progranulin was strongly correlated with serum CTRP-3 levels. CONCLUSIONS: The present study provides detailed progranulin gene expression data in sc and vis AT in a large, prospective and observational cohort of patients with severe obesity. Serum progranulin concentrations are not predicted by sc or vis AT progranulin gene expression. Thus, AT seems not to be the main source of circulating progranulin levels in obesity.

Neurobiology of the major psychoses: a translational perspective on brain structure and function-the FOR2107 consortium.

Genetic (G) and environmental (E) factors are involved in the etiology and course of the major psychoses (MP), i.e. major depressive disorder (MDD), bipolar disorder (BD), schizoaffective disorder (SZA) and schizophrenia (SZ). The neurobiological correlates by which these predispositions exert their influence on brain structure, function and course of illness are poorly understood. In the FOR2107 consortium, animal models and humans are investigated. A human cohort of MP patients, healthy subjects at genetic and/or environmental risk, and control subjects (N = 2500) has been established. Participants are followed up after 2 years and twice underwent extensive deep phenotyping (MR imaging, clinical course, neuropsychology, personality, risk/protective factors, biomaterials: blood, stool, urine, hair, saliva). Methods for data reduction, quality assurance for longitudinal MRI data, and (deep) machine learning techniques are employed. In the parallelised animal cluster, genetic risk was introduced by a rodent model (Cacna1c deficiency) and its interactions with environmental risk and protective factors are studied. The animals are deeply phenotyped regarding cognition, emotion, and social function, paralleling the variables assessed in humans. A set of innovative experimental projects connect and integrate data from the human and animal parts, investigating the role of microRNA, neuroplasticity, immune signatures, (epi-)genetics and gene expression. Biomaterial from humans and animals are analyzed in parallel. The FOR2107 consortium will delineate pathophysiological entities with common neurobiological underpinnings ("biotypes") and pave the way for an etiologic understanding of the MP, potentially leading to their prevention, the prediction of individual disease courses, and novel therapies in the future.

Evidence of an anti-inflammatory toll-like receptor 9 (TLR 9) pathway in adipocytes.

Adipocytes express various pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) and actively participate in anti-bacterial and anti-viral host defence. Obesity is associated with adipose tissue PRR expression. The potential role of Toll-like receptor 9 (TLR9) in adipocytes has not yet been investigated. Here, we evaluated TLR9 expression during adipocyte differentiation (AD) of 3T3-L1 adipocytes, in primary murine adipocytes and in different murine and human adipose tissue depots by real-time PCR, immunocytochemistry and immunohistochemistry. TLR9 expression was inhibited using specific siRNA-mediated knockdown, and TLR9 signaling was induced using specific class A, B and C agonistic CpG-oligodeoxynucleotide (ODN) treatment vs ODN controls in 3T3-L1 adipocytes and in primary murine adipocytes from Tlr9wt/wt vs Tlr9-/- mice. We found that TLR9 gene expression is induced during AD and that TLR9 protein is expressed in murine gonadal and human visceral adipocytes. AD depends on intact TLR9 expression. Tlr9-/- mice demonstrate significantly reduced adiponectin serum levels, while siRNA-mediated TLR9 knockdown led to reduced adiponectin mRNA expression in adipocytes. TLR9 ligands (CpG-ODNs) inhibit pro-inflammatory resistin secretion in mature 3T3-L1 adipocytes. Tlr9-/- as compared to Tlr9wt/wt adipocytes exhibit increased resistin and MCP1 secretion and reduced adiponectin secretion into cell culture supernatants, while TLR9 ligands (ODNs) show differential effects in Tlr9-/- vs Tlr9wt/wt primary murine adipocytes. TLR9 expression is significantly increased in visceral compared to subcutaneous adipose tissue depots in non-diabetic obese patients and correlates with systemic resistin levels in a compartment-specific manner. Thus, adipocytic TLR9 is a putative, new protective factor during (obesity-associated) adipose tissue inflammation.

A set of conformationally well-defined L/D-peptide epitopes provides a serological bar code for autoantibody subtypes.

Which conformational parameters lead to an antibody-affine peptide antigen? And in how many different conformations can we actually present the respective conformational epitope? To provide answers from a chemical point of view, we direct the bending and tethering of peptide backbones by the utilisation of a hydrophobic cluster, disulfides, and d-amino acids. Each mutation is employed pairwise on directly opposite sides of a ß-hairpin. In combination, these synthetic modules guide the formation of complementary ß-sheet-like structures, whereby the oppositely configured (l/d-)bi-disulfide pairs form with high regioselectivity. The conformational properties of the peptides are assessed by NMR spectroscopy and correlated with their antibody affinity in ELISA. From a pool of thus designed peptide antigens with distinctive complementary affinities against known rheumatoid arthritis (RA) autoantibodies, we select a set of epitopes for an immunoassay with sera of RA patients. We want to put emphasis on the idea, that the different conformational properties of the chosen antigens, containing the same epitope sequence, are mirrored in the distribution of autoantibody subtypes (or of the antibody polyclonality, respectively). Such directly comparable information can only be delivered by a set of peptides, rather than a single one. The hairpin-restriction technology of l/d-configured bi-disulfide amino acid pairs is not limited to RA but applicable to other shape-persistent hairpin motifs which are supposed to identify subgroups of protein receptors.