Inflammatory biotype of ADHD is linked to chronic stress: a data-driven analysis of the inflammatory proteome.

The association between Attention Deficit Hyperactivity Disorder (ADHD) and low-grade inflammation has been explored in children but rarely in adults. Inflammation is characteristic of some, but not all, patients with ADHD and might be influenced by ADHD medication but also lifestyle factors including nutrition, smoking, and stress. It is also still unclear if any specific symptoms are related to inflammation. Therefore, we assessed 96 inflammatory proteins in a deeply phenotyped cohort of 126 adult ADHD participants with a stable medication status using OLINK technology. A data-based, unsupervised hierarchical clustering method could identify two distinct biotypes within the 126 ADHD participants based on their inflammatory profile: a higher inflammatory potential (HIP) and a lower inflammatory protein potential (LIP) group. Biological processes that differed strongest between groups were related to the NF-κB pathway, chemokine signaling, IL-17 signaling, metabolic alterations, and chemokine attraction. A comparison of sample characteristics revealed that the HIP group was more likely to have higher levels of chronic stress (p < 0.001), a higher clinical global impression scale score (p = 0.030), and a higher risk for suicide (p = 0.032). Medication status did not influence protein levels significantly (p ≥ 0.074), but psychotropic co-medication (p ≤ 0.009) did. In conclusion, our data suggest the presence of two distinct biotypes in adults with ADHD. Higher levels of inflammatory proteins in ADHD are linked to higher levels of chronic perceived stress in a linear fashion. Further research on inflammation in adults with ADHD should take stress levels into account.

Omics and Multi-Omics Analysis for the Early Identification and Improved Outcome of Patients with Psoriatic Arthritis.

The definitive diagnosis and early treatment of many immune-mediated inflammatory diseases (IMIDs) is hindered by variable and overlapping clinical manifestations. Psoriatic arthritis (PsA), which develops in ~30% of people with psoriasis, is a key example. This mixed-pattern IMID is apparent in entheseal and synovial musculoskeletal structures, but a definitive diagnosis often can only be made by clinical experts or when an extensive progressive disease state is apparent. As with other IMIDs, the detection of multimodal molecular biomarkers offers some hope for the early diagnosis of PsA and the initiation of effective management and treatment strategies. However, specific biomarkers are not yet available for PsA. The assessment of new markers by genomic and epigenomic profiling, or the analysis of blood and synovial fluid/tissue samples using proteomics, metabolomics and lipidomics, provides hope that complex molecular biomarker profiles could be developed to diagnose PsA. Importantly, the integration of these markers with high-throughput histology, imaging and standardized clinical assessment data provides an important opportunity to develop molecular profiles that could improve the diagnosis of PsA, predict its occurrence in cohorts of individuals with psoriasis, differentiate PsA from other IMIDs, and improve therapeutic responses. In this review, we consider the technologies that are currently deployed in the EU IMI2 project HIPPOCRATES to define biomarker profiles specific for PsA and discuss the advantages of combining multi-omics data to improve the outcome of PsA patients.

The Roles of Long-Term Hyperhomocysteinemia and Micronutrient Supplementation in the AppNL-G-F Model of Alzheimer's Disease.

A causal contribution of hyperhomocysteinemia to cognitive decline and Alzheimer's disease (AD), as well as potential prevention or mitigation of the pathology by dietary intervention, have frequently been subjects of controversy. In the present in vivo study, we attempted to further elucidate the impact of elevated homocysteine (HCys) and homocysteic acid (HCA) levels, induced by dietary B-vitamin deficiency, and micronutrient supplementation on AD-like pathology, which was simulated using the amyloid-based AppNL-G-F knock-in mouse model. For this purpose, cognitive assessment was complemented by analyses of ex vivo parameters in whole blood, serum, CSF, and brain tissues from the mice. Furthermore, neurotoxicity of HCys and HCA was assessed in a separate in vitro assay. In confirmation of our previous study, older AppNL-G-F mice also exhibited subtle phenotypic impairment and extensive cerebral amyloidosis, whereas dietary manipulations did not result in significant effects. As revealed by proximity extension assay-based proteome analysis, the AppNL-G-F genotype led to an upregulation of AD-characteristic neuronal markers. Hyperhomocysteinemia, in contrast, indicated mainly vascular effects. Overall, since there was an absence of a distinct phenotype despite both a significant amyloid-ß burden and serum HCys elevation, the results in this study did not corroborate the pathological role of amyloid-ß according to the "amyloid hypothesis," nor of hyperhomocysteinemia on cognitive performance. Nevertheless, this study aided in further characterizing the AppNL-G-F model and in elucidating the role of HCys in diverse biological processes. The idea of AD prevention with the investigated micronutrients, however, was not supported, at least in this mouse model of the disease.

High-Throughput Screening for CEBPD-Modulating Compounds in THP-1-Derived Reporter Macrophages Identifies Anti-Inflammatory HDAC and BET Inhibitors.

This study aimed to identify alternative anti-inflammatory compounds that modulate the activity of a relevant transcription factor, CCAAT/enhancer binding protein delta (C/EBPδ). C/EBPδ is a master regulator of inflammatory responses in macrophages (Mϕ) and is mainly regulated at the level of CEBPD gene transcription initiation. To screen for CEBPD-modulating compounds, we generated a THP-1-derived reporter cell line stably expressing secreted alkaline phosphatase (SEAP) under control of the defined CEBPD promoter (CEBPD::SEAP). A high-throughput screening of LOPAC®1280 and ENZO®774 libraries on LPS- and IFN-γ-activated THP-1 reporter Mϕ identified four epigenetically active hits: two bromodomain and extraterminal domain (BET) inhibitors, I-BET151 and Ro 11-1464, as well as two histone deacetylase (HDAC) inhibitors, SAHA and TSA. All four hits markedly and reproducibly upregulated SEAP secretion and CEBPD::SEAP mRNA expression, confirming screening assay reliability. Whereas BET inhibitors also upregulated the mRNA expression of the endogenous CEBPD, HDAC inhibitors completely abolished it. All hits displayed anti-inflammatory activity through the suppression of IL-6 and CCL2 gene expression. However, I-BET151 and HDAC inhibitors simultaneously upregulated the mRNA expression of pro-inflammatory IL-1ß. The modulation of CEBPD gene expression shown in this study contributes to our understanding of inflammatory responses in Mϕ and may offer an approach to therapy for inflammation-driven disorders.

THP-1 and human peripheral blood mononuclear cell-derived macrophages differ in their capacity to polarize in vitro.

Macrophages (Mφ) undergo activation to pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes in response to pathophysiologic stimuli and dysregulation of the M1-M2 balance is often associated with diseases. Therefore, studying mechanisms of macrophage polarization may reveal new drug targets. Human Mφ polarization is generally studied in primary monocyte-derived Mφ (PBMC Mφ) and THP-1-derived Mφ (THP-1 Mφ). We compared the polarization profile of THP-1 Mφ with that of PBMC Mφ to assess the alternative use of THP-1 for polarization studies. Cellular morphology, the expression profiles of 18 genes and 4 cell surface proteins, and phagocytosis capacity for apoptotic cells and S. aureus bioparticles were compared between these Mφ, activated towards M1, M2a, or M2c subsets by stimulation with LPS/IFNγ, IL-4, or IL-10, respectively, for 6h, 24h and 48h. The Mφ types are unique in morphology and basal expression of polarization marker genes, particularly CCL22, in a pre-polarized state, and were differentially sensitive to polarization stimuli. Generally, M1 markers were instantly induced and gradually decreased, while M2 markers were markedly expressed at a later time. Expression profiles of M1 markers were similar between the polarized Mφ types, but M2a cell surface markers demonstrated an IL-4-dependent upregulation only in PBMC Mφ. Polarized THP-1 Mφ but not PBMC Mφ showed distinctive phagocytic capacity for apoptotic cells and bacterial antigens, respectively. In conclusion, our data suggest that THP-1 may be useful for performing studies involving phagocytosis and M1 polarization, rather than M2 polarization.

Alternative exon usage creates novel transcript variants of tumor suppressor SHREW-1 gene with differential tissue expression profile.

Shrew-1, also called AJAP1, is a transmembrane protein associated with E-cadherin-mediated adherence junctions and a putative tumor suppressor. Apart from its interaction with ß-catenin and involvement in E-cadherin internalization, little structure or function information exists. Here we explored shrew-1 expression during postnatal differentiation of mammary gland as a model system. Immunohistological analyses with antibodies against either the extracellular or the cytoplasmic domains of shrew-1 consistently revealed the expression of full-length shrew-1 in myoepithelial cells, but only part of it in luminal cells. While shrew-1 localization remained unaltered in myoepithelial cells, nuclear localization occurred in luminal cells during lactation. Based on these observations, we identified two unknown shrew-1 transcript variants encoding N-terminally truncated proteins. The smallest shrew-1 protein lacks the extracellular domain and is most likely the only variant present in luminal cells. RNA analyses of human tissues confirmed that the novel transcript variants of shrew-1 exist in vivo and exhibit a differential tissue expression profile. We conclude that our findings are essential for the understanding and interpretation of future functional and interactome analyses of shrew-1 variants.

High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion.

DNA methylation is a major regulatory process of gene transcription, and aberrant DNA methylation is associated with various diseases including cancer. Many compounds have been reported to modify DNA methylation states. Despite increasing interest in the clinical application of drugs with epigenetic effects, and the use of diagnostic markers for genome-wide hypomethylation in cancer, large-scale screening systems to measure the effects of drugs on DNA methylation are limited. In this study, we improved the previously established fluorescence polarization-based global DNA methylation assay so that it is more suitable for application to human genomic DNA. Our methyl-sensitive fluorescence polarization (MSFP) assay was highly repeatable (inter-assay coefficient of variation = 1.5%) and accurate (r2 = 0.99). According to signal linearity, only 50-80 ng human genomic DNA per reaction was necessary for the 384-well format. MSFP is a simple, rapid approach as all biochemical reactions and final detection can be performed in one well in a 384-well plate without purification steps in less than 3.5 hours. Furthermore, we demonstrated a significant correlation between MSFP and the LINE-1 pyrosequencing assay, a widely used global DNA methylation assay. MSFP can be applied for the pre-screening of compounds that influence global DNA methylation states and also for the diagnosis of certain types of cancer.

Disagreement between two common biomarkers of global DNA methylation.

BACKGROUND: The quantification of global DNA methylation has been established in epigenetic screening. As more practicable alternatives to the HPLC-based gold standard, the methylation analysis of CpG islands in repeatable elements (LINE-1) and the luminometric methylation assay (LUMA) of overall 5-methylcytosine content in "CCGG" recognition sites are most widely used. Both methods are applied as virtually equivalent, despite the hints that their results only partly agree. This triggered the present agreement assessments. RESULTS: Three different human cell types (cultured MCF7 and SHSY5Y cell lines treated with different chemical modulators of DNA methylation and whole blood drawn from pain patients and healthy volunteers) were submitted to the global DNA methylation assays employing LINE-1 or LUMA-based pyrosequencing measurements. The agreement between the two bioassays was assessed using generally accepted approaches to the statistics for laboratory method comparison studies. Although global DNA methylation levels measured by the two methods correlated, five different lines of statistical evidence consistently rejected the assumption of complete agreement. Specifically, a bias was observed between the two methods. In addition, both the magnitude and direction of bias were tissue-dependent. Interassay differences could be grouped based on Bayesian statistics, and these groups allowed in turn to re-identify the originating tissue. CONCLUSIONS: Although providing partly correlated measurements of DNA methylation, interchangeability of the quantitative results obtained with LINE-1 and LUMA was jeopardized by a consistent bias between the results. Moreover, the present analyses strongly indicate a tissue specificity of the differences between the two methods.

Domain organization of long signal peptides of single-pass integral membrane proteins reveals multiple functional capacity.

Targeting signals direct proteins to their extra- or intracellular destination such as the plasma membrane or cellular organelles. Here we investigated the structure and function of exceptionally long signal peptides encompassing at least 40 amino acid residues. We discovered a two-domain organization ("NtraC model") in many long signals from vertebrate precursor proteins. Accordingly, long signal peptides may contain an N-terminal domain (N-domain) and a C-terminal domain (C-domain) with different signal or targeting capabilities, separable by a presumably turn-rich transition area (tra). Individual domain functions were probed by cellular targeting experiments with fusion proteins containing parts of the long signal peptide of human membrane protein shrew-1 and secreted alkaline phosphatase as a reporter protein. As predicted, the N-domain of the fusion protein alone was shown to act as a mitochondrial targeting signal, whereas the C-domain alone functions as an export signal. Selective disruption of the transition area in the signal peptide impairs the export efficiency of the reporter protein. Altogether, the results of cellular targeting studies provide a proof-of-principle for our NtraC model and highlight the particular functional importance of the predicted transition area, which critically affects the rate of protein export. In conclusion, the NtraC approach enables the systematic detection and prediction of cryptic targeting signals present in one coherent sequence, and provides a structurally motivated basis for decoding the functional complexity of long protein targeting signals.