Prevalence of Autism Spectrum Disorder and Co-Morbidities in Children and Adolescents: A Systematic Literature Review.

Objective: Individuals with autism spectrum disorder often present somatic and/or psychiatric co-morbid disorders. The DSM-5 allows for consideration of additional diagnoses besides ASD and may have impacted the prevalence of co-morbidities as well as being limited in capturing the true differences in prevalence observed between males and females. We describe the prevalence of ASD and frequently observed co-morbidities in children and adolescents (<18 years) in the United States and five European countries. Methods: Two systematic literature reviews were conducted in PubMed and Embase for the period 2014-2019 and focusing on the prevalence of ASD and nine co-morbidities of interest based on their frequency and/or severity: Attention Deficit Hyperactivity Disorder (ADHD), anxiety, depressive disorders, epilepsy, intellectual disability (ID), sleep disorders, sight/hearing impairment/loss, and gastro-intestinal syndromes (GI). Results: Thirteen studies on prevalence of ASD and 33 on prevalence of co-morbidities were included. Prevalence of ASD was 1.70 and 1.85% in U.S children aged 4 and 8 years respectively, while prevalence in Europe ranged between 0.38 and 1.55%. Additionally, current evidence is supportive of a global increase in ASD prevalence over the past years. Substantial heterogeneity in prevalence of co-morbidities was observed: ADHD (0.00-86.00%), anxiety (0.00-82.20%), depressive disorders (0.00-74.80%), epilepsy (2.80-77.50%), ID (0.00-91.70%), sleep disorders (2.08-72.50%), sight/hearing impairment/loss (0.00-14.90%/0.00-4.90%), and GI syndromes (0.00-67.80%). Studies were heterogeneous in terms of design and method to estimate prevalence. Gender appears to represent a risk factor for co-morbid ADHD (higher in males) and epilepsy/seizure (higher in females) while age is also associated with ADHD and anxiety (increasing until adolescence). Conclusion: Our results provide a descriptive review of the prevalence of ASD and its co-morbidities in children and adolescents. These insights can be valuable for clinicians and parents/guardians of autistic children. Prevalence of ASD has increased over time while co-morbidities bring additional heterogeneity to the clinical presentation, which further advocates for personalized approaches to treatment and support. Having a clear understanding of the prevalence of ASD and its co-morbidities is important to raise awareness among stakeholders.Appeared originally in Front Psychiatry 2021; 12:744709.

Transcriptomic characterization of the human segmental endotoxin challenge model.

Segmental instillation of lipopolysaccharide (LPS) by bronchoscopy safely induces transient airway inflammation in human lungs. This model enables investigation of pulmonary inflammatory mechanisms as well as pharmacodynamic analysis of investigational drugs. The aim of this work was to describe the transcriptomic profile of human segmental LPS challenge with contextualization to major respiratory diseases. Pre-challenge bronchoalveolar lavage (BAL) fluid and biopsies were sampled from 28 smoking, healthy participants, followed by segmental instillation of LPS and saline as control. Twenty-four hours post instillation, BAL and biopsies were collected from challenged lung segments. Total RNA of cells from BAL and biopsy samples were sequenced and analysed for differentially expressed genes (DEGs). After challenge with LPS compared with saline, 6316 DEGs were upregulated and 241 were downregulated in BAL, but only one DEG was downregulated in biopsy samples. Upregulated DEGs in BAL were related to molecular functions such as "Inflammatory response" or "chemokine receptor activity", and upregulated pro-inflammatory pathways such as "Wnt-"/"Ras-"/"JAK-STAT" "-signaling pathway". Furthermore, the segmental LPS challenge model resembled aspects of the five most prevalent respiratory diseases chronic obstructive pulmonary disease (COPD), asthma, pneumonia, tuberculosis and lung cancer and featured similarities with acute exacerbations in COPD (AECOPD) and community-acquired pneumonia. Overall, our study provides extensive information about the transcriptomic profile from BAL cells and mucosal biopsies following LPS challenge in healthy smokers. It expands the knowledge about the LPS challenge model providing potential overlap with respiratory diseases in general and infection-triggered respiratory insults such as AECOPD in particular.

nf-core/nanostring: a pipeline for reproducible NanoString nCounter analysis.

MOTIVATION: The NanoString™ nCounter® technology platform is a widely used targeted quantification platform for the analysis of gene expression of up to ∼800 genes. Whereas the software tools by the manufacturer can perform the analysis in an interactive and GUI driven approach, there is no portable and user-friendly workflow available that can be used to perform reproducible analysis of multiple samples simultaneously in a scalable fashion on different computing infrastructures. RESULTS: Here, we present the nf-core/nanostring open-source pipeline to perform a comprehensive analysis including quality control and additional features such as expression visualization, annotation with additional metadata and input creation for differential gene expression analysis. The workflow features an easy installation, comprehensive documentation, open-source code with the possibility for further extensions, a strong portability across multiple computing environments and detailed quality metrics reporting covering all parts of the pipeline. nf-core/nanostring has been implemented in the Nextflow workflow language and supports Docker, Singularity, Podman container technologies as well as Conda environments, enabling easy deployment on any Nextflow supported compatible system, including most widely used cloud computing environments such as Google GCP or Amazon AWS. AVAILABILITY AND IMPLEMENTATION: The source code, documentation and installation instructions as well as results for continuous tests are freely available at https://github.com/nf-core/nanostring and https://nf-co.re/nanostring.

Harnessing Wearable Devices for Emotional Intelligence: Therapeutic Applications in Digital Health.

Emotional intelligence strives to bridge the gap between human and machine interactions. The application of such systems varies and is becoming more prominent as healthcare services seek to provide more efficient care by utilizing smart digital health apps. One application in digital health is the incorporation of emotion recognition systems as a tool for therapeutic interventions. To this end, a system is designed to collect and analyze physiological signal data, such as electrodermal activity (EDA) and electrocardiogram (ECG), from smart wearable devices. The data are collected from different subjects of varying ages taking part in a study on emotion induction methods. The obtained signals are processed to identify stimulus trigger instances and classify the different reaction stages, as well as arousal strength, using signal processing and machine learning techniques. The reaction stages are identified using a support vector machine algorithm, while the arousal strength is classified using the ResNet50 network architecture. The findings indicate that the EDA signal effectively identifies the emotional trigger, registering a root mean squared error (RMSE) of 0.9871. The features collected from the ECG signal show efficient emotion detection with 94.19% accuracy. However, arousal strength classification is only able to reach 60.37% accuracy on the given dataset. The proposed system effectively detects emotional reactions and can categorize their arousal strength in response to specific stimuli. Such a system could be integrated into therapeutic settings to monitor patients' emotional responses during therapy sessions. This real-time feedback can guide therapists in adjusting their strategies or interventions.

Integrative multi-omics analysis reveals novel idiopathic pulmonary fibrosis endotypes associated with disease progression.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is characterized by the accumulation of extracellular matrix in the pulmonary interstitium and progressive functional decline. We hypothesized that integration of multi-omics data would identify clinically meaningful molecular endotypes of IPF. METHODS: The IPF-PRO Registry is a prospective registry of patients with IPF. Proteomic and transcriptomic (including total RNA [toRNA] and microRNA [miRNA]) analyses were performed using blood collected at enrollment. Molecular data were integrated using Similarity Network Fusion, followed by unsupervised spectral clustering to identify molecular subtypes. Cox proportional hazards models tested the relationship between these subtypes and progression-free and transplant-free survival. The molecular subtypes were compared to risk groups based on a previously described 52-gene (toRNA expression) signature. Biological characteristics of the molecular subtypes were evaluated via linear regression differential expression and canonical pathways (Ingenuity Pathway Analysis [IPA]) over-representation analyses. RESULTS: Among 232 subjects, two molecular subtypes were identified. Subtype 1 (n = 105, 45.3%) and Subtype 2 (n = 127, 54.7%) had similar distributions of age (70.1 +/- 8.1 vs. 69.3 +/- 7.6 years; p = 0.31) and sex (79.1% vs. 70.1% males, p = 0.16). Subtype 1 had more severe disease based on composite physiologic index (CPI) (55.8 vs. 51.2; p = 0.002). After adjusting for CPI and antifibrotic treatment at enrollment, subtype 1 experienced shorter progression-free survival (HR 1.79, 95% CI 1.28,2.56; p = 0.0008) and similar transplant-free survival (HR 1.30, 95% CI 0.87,1.96; p = 0.20) as subtype 2. There was little agreement in the distribution of subjects to the molecular subtypes and the risk groups based on 52-gene signature (kappa = 0.04, 95% CI= -0.08, 0.17), and the 52-gene signature risk groups were associated with differences in transplant-free but not progression-free survival. Based on heatmaps and differential expression analyses, proteins and miRNAs (but not toRNA) contributed to classification of subjects to the molecular subtypes. The IPA showed enrichment in pulmonary fibrosis-relevant pathways, including mTOR, VEGF, PDGF, and B-cell receptor signaling. CONCLUSIONS: Integration of transcriptomic and proteomic data from blood enabled identification of clinically meaningful molecular endotypes of IPF. If validated, these endotypes could facilitate identification of individuals likely to experience disease progression and enrichment of clinical trials. TRIAL REGISTRATION: NCT01915511.

Pustular psoriasis: Molecular pathways and effects of spesolimab in generalized pustular psoriasis.

BACKGROUND: The IL-36 pathway plays a key role in the pathogenesis of generalized pustular psoriasis (GPP). In a proof-of-concept clinical trial, treatment with spesolimab, an anti-IL-36 receptor antibody, resulted in rapid skin and pustular clearance in patients presenting with GPP flares. OBJECTIVE: We sought to compare the molecular profiles of lesional and nonlesional skin from patients with GPP or palmoplantar pustulosis (PPP) with skin from healthy volunteers, and to investigate the molecular changes after spesolimab treatment in the skin and blood of patients with GPP flares. METHODS: Pre- and post-treatment skin and blood samples were collected from patients with GPP who participated in a single-arm, phase I study (n = 7). Skin biopsies from patients with PPP (n = 8) and healthy volunteers (n = 16) were obtained for comparison at baseline. Biomarkers were assessed by RNA-sequencing, histopathology, and immunohistochemistry. RESULTS: In GPP and PPP lesions, 1287 transcripts were commonly upregulated or downregulated. Selected transcripts from the IL-36 signaling pathway were upregulated in untreated GPP and PPP lesions. In patients with GPP, IL-36 pathway-related signatures, TH1/TH17 and innate inflammation signaling, neutrophilic mediators, and keratinocyte-driven inflammation pathways were downregulated by spesolimab as early as week 1. Spesolimab also decreased related serum biomarkers and cell populations in the skin lesions from patients with GPP, including CD3+ T, CD11c+, and IL-36γ+ cells and lipocalin-2-expressing cells. CONCLUSIONS: In patients with GPP, spesolimab showed rapid modulation of commonly dysregulated molecular pathways in GPP and PPP, which may be associated with improved clinical outcomes.

Prevalence of Autism Spectrum Disorder and Co-morbidities in Children and Adolescents: A Systematic Literature Review.

Objective: Individuals with autism spectrum disorder often present somatic and/or psychiatric co-morbid disorders. The DSM-5 allows for consideration of additional diagnoses besides ASD and may have impacted the prevalence of co-morbidities as well as being limited in capturing the true differences in prevalence observed between males and females. We describe the prevalence of ASD and frequently observed co-morbidities in children and adolescents (<18 years) in the United States and five European countries. Methods: Two systematic literature reviews were conducted in PubMed and Embase for the period 2014-2019 and focusing on the prevalence of ASD and nine co-morbidities of interest based on their frequency and/or severity: Attention Deficit Hyperactivity Disorder (ADHD), anxiety, depressive disorders, epilepsy, intellectual disability (ID), sleep disorders, sight/hearing impairment/loss, and gastro-intestinal syndromes (GI). Results: Thirteen studies on prevalence of ASD and 33 on prevalence of co-morbidities were included. Prevalence of ASD was 1.70 and 1.85% in US children aged 4 and 8 years respectively, while prevalence in Europe ranged between 0.38 and 1.55%. Additionally, current evidence is supportive of a global increase in ASD prevalence over the past years. Substantial heterogeneity in prevalence of co-morbidities was observed: ADHD (0.00-86.00%), anxiety (0.00-82.20%), depressive disorders (0.00-74.80%), epilepsy (2.80-77.50%), ID (0.00-91.70%), sleep disorders (2.08-72.50%), sight/hearing impairment/loss (0.00-14.90%/0.00-4.90%), and GI syndromes (0.00-67.80%). Studies were heterogeneous in terms of design and method to estimate prevalence. Gender appears to represent a risk factor for co-morbid ADHD (higher in males) and epilepsy/seizure (higher in females) while age is also associated with ADHD and anxiety (increasing until adolescence). Conclusion: Our results provide a descriptive review of the prevalence of ASD and its co-morbidities in children and adolescents. These insights can be valuable for clinicians and parents/guardians of autistic children. Prevalence of ASD has increased over time while co-morbidities bring additional heterogeneity to the clinical presentation, which further advocates for personalized approaches to treatment and support. Having a clear understanding of the prevalence of ASD and its co-morbidities is important to raise awareness among stakeholders.

RORγt inhibitors block both IL-17 and IL-22 conferring a potential advantage over anti-IL-17 alone to treat severe asthma.

BACKGROUND: RORγt is a transcription factor that enables elaboration of Th17-associated cytokines (including IL-17 and IL-22) and is proposed as a pharmacological target for severe asthma. METHODS: IL-17 immunohistochemistry was performed in severe asthma bronchial biopsies (specificity confirmed with in situ hybridization). Primary human small airway epithelial cells in air liquid interface and primary bronchial smooth muscle cells were stimulated with recombinant human IL-17 and/or IL-22 and pro-inflammatory cytokines measured. Balb/c mice were challenged intratracheally with IL-17 and/or IL-22 and airway hyperreactivity, pro-inflammatory cytokines and airway neutrophilia measured. Balb/c mice were sensitized intraperitoneally and challenged intratracheally with house dust mite extract and the effect of either a RORγt inhibitor (BIX119) or an anti-IL-11 antibody assessed on airway hyperreactivity, pro-inflammatory cytokines and airway neutrophilia measured. RESULTS: We confirmed in severe asthma bronchial biopsies both the presence of IL-17-positive lymphocytes and that an IL-17 transcriptome profile in a severe asthma patient sub-population. Both IL-17 and IL-22 stimulated the release of pro-inflammatory cytokine and chemokine release from primary human lung cells and in mice. Furthermore, IL-22 in combination with IL-17, but neither alone, elicits airway hyperresponsiveness (AHR) in naïve mice. A RORγt inhibitor specifically blocked both IL-17 and IL-22, AHR and neutrophilia in a mouse house dust mite model unlike other registered or advanced pipeline modes of action. Full efficacy versus these parameters was associated with 90% inhibition of IL-17 and 50% inhibition of IL-22. In contrast, anti-IL-17 also blocked IL-17, but not IL-22, AHR or neutrophilia. Moreover, the deregulated genes in the lungs from these mice correlated well with deregulated genes from severe asthma biopsies suggesting that this model recapitulates significant severe asthma-relevant biology. Furthermore, these genes were reversed upon RORγt inhibition in the HDM model. Cell deconvolution suggested that the responsible cells were corticosteroid insensitive γδ-T-cells. CONCLUSION: These data strongly suggest that both IL-17 and IL-22 are required for Th2-low endotype associated biology and that a RORγt inhibitor may provide improved clinical benefit in a severe asthma sub-population of patients by blocking both IL-17 and IL-22 biology compared with blocking IL-17 alone.

Association of Consensus Molecular Subtypes and Molecular Markers With Clinical Outcomes in Patients With Metastatic Colorectal Cancer: Biomarker Analyses From LUME-Colon 1.

INTRODUCTION: LUME-Colon 1 (NCT02149108) was a global, placebo-controlled phase III study of nintedanib in advanced colorectal cancer (CRC). Pre-specified biomarker analyses investigated the association of CRC consensus molecular subtypes (CMS) and tumor genomic and circulating biomarkers with clinical outcomes. MATERIALS AND METHODS: Archival tumor tissue, cell-free DNA (cfDNA), and plasma samples were collected for genomic, transcriptomic, and proteomic analyses to investigate potential associations between CRC CMS and other biomarkers with nintedanib response and clinical outcomes. RESULTS: Of the 765 treated patients, 735, 245, and 192 patient samples were analyzed in the circulating protein, tumor tissue, and cfDNA datasets, respectively. Patients were classified as CMS1 (1.7%), CMS2 (27.7%), CMS3 (0.9%), CMS4 (51.5%), or unclassified (18.2%). Unclassified/mixed CMS was associated with longer overall survival (OS) with nintedanib vs. CMS2 or CMS4 (interaction P-value = .0086); no association was observed for CMS4. Gene expression-based pathway analysis revealed an association between vascular endothelial growth factor-related signaling and OS for nintedanib (P = .0498). The most frequently detected somatic mutations were APC (72.0% [tumor tissue] vs. 56.8% [cfDNA]), TP53 (47.1% vs. 34.9%), KRAS (40.8% vs. 28.6%), and PIK3CA (16.6% vs. 11.5%); concordance rates were > 80%. Median OS differences were observed for APC and TP53 mutations vs. wild-type in cfDNA, indicating a potential prognostic value. Circulating ANG-2, CA-9, CEACAM1, collagen-IV, IGFBP-1, ICAM-1, IL-8, and uPAR were potentially prognostic for both OS and progression-free survival. CONCLUSION: We demonstrated the feasibility of large-scale biomarker analyses and CMS classification within a global clinical trial, and identified signals suggesting a potential for greater nintedanib treatment response in the unclassified/mixed CMS subgroup, despite these tumors showing heterogeneous patterns of CMS mixtures. Our results revealed a high degree of concordance in somatic mutations between tumor tissue and cfDNA. Associations with prognosis for cfDNA somatic mutations, as well as several protein-based biomarkers, may warrant further investigation in future trials.

Fecal MicroRNAs Show Promise as Noninvasive Crohn's Disease Biomarkers.

BACKGROUND: Short non-coding microRNAs (miRNAs) are involved in various cellular processes during disease progression of Crohn's disease (CD) and remarkably stable in feces, which make them attractive biomarker candidates for reflecting intestinal inflammatory processes. Here we investigated the potential of fecal miRNAs as noninvasive and translational CD biomarkers. METHODS: MiRNAs were screened in feces of 52 patients with CD and 15 healthy controls using RNA sequencing and the results were confirmed by PCR. The relationship between fecal miRNA levels and the clinical CD activity index (CDAI) or CD endoscopic index of severity (CDEIS) was explored, respectively. Additionally, fecal miRNAs were investigated in dextran sodium sulfate, adoptive T-cell transfer, and Helicobacter typhlonius/stress-induced murine colitis models using the NanoString platform. RESULTS: Nine miRNAs (miR-15a-5p, miR-16-5p, miR-128-3p, miR-142-5p, miR-24-3p, miR-27a-3p, miR-223-3p, miR-223-5p, and miR-3074-5p) were significantly (adj. P < 0.05, >3-fold) increased whereas 8 miRNAs (miR-10a-5p, miR-10b-5p, miR-141-3p, miR-192-5p, miR-200a-3p, miR-375, miR-378a-3p, and let-7g-5p) were significantly decreased in CD. MiR-192-5p, miR-375, and miR-141-3p correlated (P < 0.05) with both CDAI and CDEIS whereas miR-15a-5p correlated only with CDEIS. Deregulated expression of miR-223-3p, miR-16-5p, miR-15a-5p, miR-24-3p, and miR-200a-3p was also observed in murine models. The identified altered fecal miRNA levels reflect pathophysiological mechanisms in CD, such as Th1 and Th17 inflammation, autophagy, and fibrotic processes. CONCLUSIONS: Our translational study assessed global fecal miRNA changes of patients with CD and relevant preclinical models. These fecal miRNAs show promise as translational and clinically useful noninvasive biomarkers for mechanistic investigation of intestinal pathophysiology, including monitoring of disease progression.

Exosomal miRNAs as Potential Biomarkers to Monitor Phosphodiesterase 5 Inhibitor Induced Anti-Fibrotic Effects on CCl4 Treated Rats.

MicroRNAs (miRNAs) are short, non-coding RNA species that are important post-transcriptional regulators of gene expression and play an important role in the pathogenesis of non-alcoholic fatty liver disease. Here, we investigated the phosphodiesterase 5 (PDE5) inhibitor induced effects on hepatic and plasma exosomal miRNA expression in CCl4-treated rats. In the present study, hepatic miRNA profiling was conducted using the Nanostring nCounter technology and mRNA profiling using RNA sequencing from PDE5 treated rats in the model of CCl4-induced liver fibrosis. To evaluate if the PDE5 inhibitor affected differentially expressed miRNAs in the liver can be detected in plasma exosomes, qRT-PCR specific assays were used. In livers from CCl4-treated rats, the expression of 22 miRNAs was significantly increased (> 1.5-fold, adj. p < 0.05), whereas the expression of 16 miRNAs was significantly decreased (> 1.5-fold, adj. p < 0.05). The majority of the deregulated miRNA species are implicated in fibrotic and inflammatory processes. The PDE5 inhibitor suppressed the induction of pro-fibrotic miRNAs, such as miR-99b miR-100 and miR-199a-5p, and restored levels of anti-fibrotic miR-122 and miR-192 in the liver. In plasma exosomes, we observed elevated levels of miR-99b, miR-100 and miR-142-3p after treatment with the PDE5-inhibitor compared to CCl4/Vehicle-treated. Our study demonstrated for the first time that during the development of hepatic fibrosis in the preclinical model of CCl4-induced liver fibrosis, defined aspects of miRNA regulated liver pathogenesis are influenced by PDE5 treatment. In conclusion, miRNA profiling of plasma exosomes might be used as a biomarker for NASH progression and monitoring of treatment effects.

Integrated microRNA/mRNA expression profiling of the skin of psoriasis patients.

BACKGROUND: Psoriasis is a chronic inflammatory disease characterized by demarcated, raised, and scaling skin lesions. It often serves as a model for immune-mediated disorders. Gene expression profiling of affected skin has allowed insights into psoriasis pathogenesis. However, the mechanisms leading to specific mRNA expression alterations in psoriasis are barely understood. OBJECTIVES: To perform integrated microRNA-mRNA expression studies of non-lesional, peri-lesional, and lesional skin from psoriasis patients. METHODS: Cutaneous microRNA and mRNA expression profiles of 14 patients using Nanostring nCounter-technology and RNA sequencing as well as in vitro keratinocyte stimulation and qPCR studies. RESULTS: Only 3.5 % of microRNAs manifested a robust gradual expression trend from non-lesional to paired lesional skin, with 61 % being upregulated and 39 % being downregulated. Relevance of these microRNA regulations was supported by their inverse association with 57 % of the mRNA species found to be regulated during psoriatic lesion development. Many of the involved mRNAs were downregulated and functionally related to keratinocyte metabolism, barrier function, and neuronal signaling, and were already regulated in peri-lesional skin. An integrated correlation analysis revealed a robust interaction for 134 microRNAs/mRNAs pairs. In vitro keratinocyte studies of selected microRNAs/mRNAs revealed regulations of all analyzed microRNAs in a psoriasis-like manner by IL-17A/TNF-α (e.g. hsa-miR-23a-3p), IFN-γ (e.g. hsa-miR-106a-5p/miR-17-5p), or IL-24 (e.g. hsa-miR-203a-3p). Moreover, most of their predicted target mRNAs (e.g. ID4, EPHB2) were respectively altered by the same cytokines. CONCLUSION: Our study suggests that, during development of psoriatic lesions, defined aspects of psoriasis pathogenesis are regulated by the action of microRNAs.

Peripheral blood proteomic profiling of idiopathic pulmonary fibrosis biomarkers in the multicentre IPF-PRO Registry.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease for which diagnosis and management remain challenging. Defining the circulating proteome in IPF may identify targets for biomarker development. We sought to quantify the circulating proteome in IPF, determine differential protein expression between subjects with IPF and controls, and examine relationships between protein expression and markers of disease severity. METHODS: This study involved 300 patients with IPF from the IPF-PRO Registry and 100 participants without known lung disease. Plasma collected at enrolment was analysed using aptamer-based proteomics (1305 proteins). Linear regression was used to determine differential protein expression between participants with IPF and controls and associations between protein expression and disease severity measures (percent predicted values for forced vital capacity [FVC] and diffusion capacity of the lung for carbon monoxide [DLco]; composite physiologic index [CPI]). Multivariable models were fit to select proteins that best distinguished IPF from controls. RESULTS: Five hundred fifty one proteins had significantly different levels between IPF and controls, of which 47 showed a |log2(fold-change)| > 0.585 (i.e. > 1.5-fold difference). Among the proteins with the greatest difference in levels in patients with IPF versus controls were the glycoproteins thrombospondin 1 and von Willebrand factor and immune-related proteins C-C motif chemokine ligand 17 and bactericidal permeability-increasing protein. Multivariable classification modelling identified nine proteins that, when considered together, distinguished IPF versus control status with high accuracy (area under receiver operating curve = 0.99). Among participants with IPF, 14 proteins were significantly associated with FVC % predicted, 23 with DLco % predicted, 14 with CPI. Four proteins (roundabout homolog-2, spondin-1, polymeric immunoglobulin receptor, intercellular adhesion molecule 5) demonstrated the expected relationship across all three disease severity measures. When considered in pathways analyses, proteins associated with the presence or severity of IPF were enriched in pathways involved in platelet and haemostatic responses, vascular or platelet derived growth factor signalling, immune activation, and extracellular matrix organisation. CONCLUSIONS: Patients with IPF have a distinct circulating proteome and can be distinguished using a nine-protein profile. Several proteins strongly associate with disease severity. The proteins identified may represent biomarker candidates and implicate pathways for further investigation. TRIAL REGISTRATION: ClinicalTrials.gov (NCT01915511).

Psoriatic skin molecular and histopathologic profiles after treatment with risankizumab versus ustekinumab.

BACKGROUND: IL-23 contributes to the activation, maintenance, and proliferation of TH17 cells and plays a major role in psoriasis pathophysiology. IL-23p19 inhibition with risankizumab resulted in superior clinical responses in patients with psoriasis compared with ustekinumab (dual IL-12/IL-23 inhibitor), but comparative molecular effects have not been established. OBJECTIVE: We investigated the similarities and differences in molecular and histopathologic profiles in skin lesions from patients with psoriasis receiving risankizumab versus ustekinumab at an early time point. METHODS: Lesional skin biopsy samples from 81 patients with moderate-to-severe plaque psoriasis participating in 2 different studies (a phase I risankizumab study and a phase II study of risankizumab vs ustekinumab) were analyzed by using histopathology, immunohistochemistry, and RNA sequencing. RESULTS: Risankizumab induced a rapid decrease in levels of proteins and transcriptomic biomarkers associated with the IL-23 pathway, which were maintained through 8 weeks. At week 4, risankizumab decreased histopathologic expression of biomarkers, including K16, Ki67, CD3, lipocalin-2, CD11c, dendritic cell lysosome-associated membrane glycoprotein, ß-defensin 2, and S100A7; global histopathologic scoring revealed that 54% and 69% of patients treated with 90 or 180 mg of risankizumab, respectively, were graded as experiencing "excellent improvement" versus 29% of patients treated with ustekinumab. At week 4, there was a common decrease in expression of 2645 genes expressed in lesional skin between patients receiving risankizumab and ustekinumab and a significant decrease in 2682 genes unique to risankizumab treatment. Risankizumab more strongly downregulated expression of genes associated with keratinocytes, epidermal cells, and monocytes, versus ustekinumab. CONCLUSION: Risankizumab demonstrated more pronounced changes in the molecular and histopathologic profile of psoriatic skin lesions compared with ustekinumab at week 4.

Selective IL-23 Inhibition by Risankizumab Modulates the Molecular Profile in the Colon and Ileum of Patients With Active Crohn's Disease: Results From a Randomised Phase II Biopsy Sub-study.

BACKGROUND AND AIMS: We aimed to investigate the underlying mechanism of action of risankizumab, a monoclonal antibody targeting the IL-23 p19 subunit, previously reported to induce clinical and endoscopic remission in a randomised phase II study in patients with active Crohn's disease. METHODS: Ileum and colon biopsies obtained at screening and Week 12 from a subgroup of patients [n = 106] in the risankizumab phase II study were analysed by transcriptome-wide RNA-Seq profiling. Univariate associations were assessed using linear modelling. RESULTS: By Week 12, risankizumab significantly decreased [p < 0.005] the expression of 1880 and 765 genes in the colon [false-discovery rate = 0.02] and ileum [false-discovery rate = 0.05], respectively. These genes were associated with the IL-23/IL-17 axis, Th1 pathway, innate immunity, and tissue turnover. Colonic transcriptomic profiles following risankizumab treatment reflected the transcriptomic changes observed in patients achieving endoscopic response and remission at Week 12 and were significantly different from placebo [p < 0.005]. The colonic transcriptomic profile, significantly modulated by risankizumab at Week 12, was indicative of suppression of pathways associated with epithelial biology. Furthermore, pathways associated with Crohn's disease modulated by risankizumab treatment included second messenger-mediated signalling, immune response, lymphocyte and leucocyte activation, lymphocyte differentiation and cell-cell adhesion. CONCLUSIONS: Endoscopic remission and response observed with risankizumab in patients with active Crohn's disease was associated with significant transcriptomic changes in the colon, compared with placebo. Differentiated expression of genes associated with the IL-23/IL-17 axis was observed in the colon and ileum 12 weeks after risankizumab treatment.

Discovery and translation of a target engagement marker for AMP-activated protein kinase (AMPK).

BACKGROUND: Activation of the AMP-activated protein kinase (AMPK) is an attractive approach for the treatment of type 2 diabetes. AMPK activation reduces glucose levels in animal models of type 2 diabetes by increasing glucose uptake in skeletal muscles and reducing hepatic glucose production. Furthermore, AMPK activation ameliorates hepatic steatosis in animal models. For the clinical development of AMPK activators it is essential to have a reliable target engagement marker for appropriate dose finding and to support proof of clinical principle. While the activation of AMPK by quantification of the phosphorylation of AMPK at Thr172 in target tissues can be assessed pre-clinically, this is not feasible in clinical studies. Therefore, we attempted to identify and translate a peripheral target engagement biomarker downstream of AMPK activation for clinical use in blood samples. METHODS: For pharmacological activation of AMPK, two AMPK activators were synthesized (compound 1 and 2). A compound with structural similarities but no pharmacological effect on AMPK phosphorylation was synthesized as negative control (compound 3). Whole blood from healthy volunteers was incubated with an AMPK activator for up to 6 hours and mRNA sequencing was performed. Additionally, human PBMCs were isolated to evaluate Thr172-phosphorylation of AMPK in Western blots. In order to enable identification of translatable biomarker candidates, blood samples from HanWistar rats treated for two weeks with an AMPK activator were also subjected to mRNA sequencing. Furthermore, concentration-response curves for four biomarker candidates were recorded in human blood samples using Nanostring nCounter technology. Finally, ZDF rats were treated with increasing doses of compound 2 for five weeks to investigate the glucose-lowering efficacy. To investigate changes of mRNA expression of two selected biomarker candidates in this ZDF rat study, qRT-PCR was performed. RESULTS: Pharmacological activation of AMPK in human PBMCs revealed an increase in Thr172-phosphorylation of AMPK, confirming target engagement in these blood cells. RNA sequencing of human blood samples identified 608 deregulated genes after AMPK activation. Additionally, AMPK activation led to deregulation of 367 genes in whole blood from HanWistar rats which mapped to the respective human genes. 22 genes out of the intersection of genes deregulated in both species are proposed as potential translatable target engagement biomarker candidates. The most prominent genes were transmembrane glycoprotein NMB (GPNMB, osteoactivin), calcium-binding protein A9 (S100A9), peptidoglycan recognition protein (PGLYRP1) and Ras homolog gene family, member B (RHOB). Specificity for AMPK was shown by testing inactive compound 3 in HanWistar rats. The exposure-effect relationship for GPNMB was investigated in a subchronic study in diabetic ZDF rats. GPNMB showed a dose-dependent up-regulation both acutely and after subchronic dosing. GPNMB up-regulation correlated with an increased Thr172-phosphorylation of AMPK in liver and quadriceps muscle in rats. CONCLUSION: GPNMB has been identified as a translatable target engagement biomarker for use in clinical studies.

Clonal Evolutionary Analysis during HER2 Blockade in HER2-Positive Inflammatory Breast Cancer: A Phase II Open-Label Clinical Trial of Afatinib +/- Vinorelbine.

BACKGROUND: Inflammatory breast cancer (IBC) is a rare, aggressive form of breast cancer associated with HER2 amplification, with high risk of metastasis and an estimated median survival of 2.9 y. We performed an open-label, single-arm phase II clinical trial (ClinicalTrials.gov NCT01325428) to investigate the efficacy and safety of afatinib, an irreversible ErbB family inhibitor, alone and in combination with vinorelbine in patients with HER2-positive IBC. This trial included prospectively planned exome analysis before and after afatinib monotherapy. METHODS AND FINDINGS: HER2-positive IBC patients received afatinib 40 mg daily until progression, and thereafter afatinib 40 mg daily and intravenous vinorelbine 25 mg/m2 weekly. The primary endpoint was clinical benefit; secondary endpoints were objective response (OR), duration of OR, and progression-free survival (PFS). Of 26 patients treated with afatinib monotherapy, clinical benefit was achieved in 9 patients (35%), 0 of 7 trastuzumab-treated patients and 9 of 19 trastuzumab-naïve patients. Following disease progression, 10 patients received afatinib plus vinorelbine, and clinical benefit was achieved in 2 of 4 trastuzumab-treated and 0 of 6 trastuzumab-naïve patients. All patients had treatment-related adverse events (AEs). Whole-exome sequencing of tumour biopsies taken before treatment and following disease progression on afatinib monotherapy was performed to assess the mutational landscape of IBC and evolutionary trajectories during therapy. Compared to a cohort of The Cancer Genome Atlas (TCGA) patients with HER2-positive non-IBC, HER2-positive IBC patients had significantly higher mutational and neoantigenic burden, more frequent gain-of-function TP53 mutations and a recurrent 11q13.5 amplification overlapping PAK1. Planned exploratory analysis revealed that trastuzumab-naïve patients with tumours harbouring somatic activation of PI3K/Akt signalling had significantly shorter PFS compared to those without (p = 0.03). High genomic concordance between biopsies taken before and following afatinib resistance was observed with stable clonal structures in non-responding tumours, and evidence of branched evolution in 8 of 9 tumours analysed. Recruitment to the trial was terminated early following the LUX-Breast 1 trial, which showed that afatinib combined with vinorelbine had similar PFS and OR rates to trastuzumab plus vinorelbine but shorter overall survival (OS), and was less tolerable. The main limitations of this study are that the results should be interpreted with caution given the relatively small patient cohort and the potential for tumour sampling bias between pre- and post-treatment tumour biopsies. CONCLUSIONS: Afatinib, with or without vinorelbine, showed activity in trastuzumab-naïve HER2-positive IBC patients in a planned subgroup analysis. HER2-positive IBC is characterized by frequent TP53 gain-of-function mutations and a high mutational burden. The high mutational load associated with HER2-positive IBC suggests a potential role for checkpoint inhibitor therapy in this disease. TRIAL REGISTRATION: ClinicalTrials.gov NCT01325428.

Characterization of Micro-RNA Changes during the Progression of Type 2 Diabetes in Zucker Diabetic Fatty Rats.

The aim of the present pilot study was the identification of micro-RNA changes over time during the development and progression of type 2 diabetes (T2D) in Zucker diabetic fatty rats (ZDF rats). T2D is a complex metabolic disorder that is characterized, inter alia, by progressive failure of pancreatic ß cells to produce insulin, but also by functional or morphological modifications of others organ, such as liver, adipose tissue and the cardiovascular system. Micro-RNAs are a novel class of biomarkers that have the potential to represent biomarkers of disease progression. In this study, the onset and progression of diabetes was followed in ZDF rats from six weeks until 17 weeks of age. After an initial phase of hyperinsulinemia, the animals developed T2D and lost the capacity to produce sufficient insulin. Circulating miRNAs were measured from plasma samples at four time points: pre-diabetes (six weeks of age), hyperinsulinemia (eight weeks), ß cell failure (11 weeks) and late-stage diabetes (17 weeks) using TaqMan miRNA arrays. Bioinformatic analysis revealed distinct changes of circulating miRNAs over time. Several miRNAs were found to be increased over the course of the disease progression, such as miR-122, miR-133, miR-210 and miR-375. The most significantly decreased miRNAs were miR-140, miR-151-3p, miR-185, miR-203, miR-434-3p and miR-450a. Some of the miRNAs have also been identified in type 2 diabetic patients recently and, therefore, may have the potential to be useful biomarkers for the disease progression of T2D and/or the treatment response for anti-diabetic medications.

Urinary Exosomal miRNA Signature in Type II Diabetic Nephropathy Patients.

MicroRNAs (miRNAs) are short non-coding RNA species which are important post-transcriptional regulators of gene expression and play an important role in the pathogenesis of diabetic nephropathy. miRNAs are present in urine in a remarkably stable form packaged in extracellular vesicles, predominantly exosomes. In the present study, urinary exosomal miRNA profiling was conducted in urinary exosomes obtained from 8 healthy controls (C), 8 patients with type II diabetes (T2D) and 8 patients with type II diabetic nephropathy (DN) using Agilent´s miRNA microarrays. In total, the expression of 16 miRNA species was deregulated (>2-fold) in DN patients compared to healthy donors and T2D patients: the expression of 14 miRNAs (miR-320c, miR-6068, miR-1234-5p, miR-6133, miR-4270, miR-4739, miR-371b-5p, miR-638, miR-572, miR-1227-5p, miR-6126, miR-1915-5p, miR-4778-5p and miR-2861) was up-regulated whereas the expression of 2 miRNAs (miR-30d-5p and miR-30e-5p) was down-regulated. Most of the deregulated miRNAs are involved in progression of renal diseases. Deregulation of urinary exosomal miRNAs occurred in micro-albuminuric DN patients but not in normo-albuminuric DN patients. We used qRT-PCR based analysis of the most strongly up-regulated miRNAs in urinary exosomes from DN patients, miRNAs miR-320c and miR-6068. The correlation of miRNA expression and micro-albuminuria levels could be replicated in a confirmation cohort. In conclusion, urinary exosomal miRNA content is altered in type II diabetic patients with DN. Deregulated miR-320c, which might have an impact on the TGF-ß-signaling pathway via targeting thrombospondin 1 (TSP-1) shows promise as a novel candidate marker for disease progression in type II DN that should be evaluated in future studies.

The Cost-Effectiveness of Emergency Hormonal Contraception with Ulipristal Acetate versus Levonorgestrel for Minors in France.

OBJECTIVE: To evaluate the cost-effectiveness of ulipristal acetate and levonorgestrel in minors in France, and analyze whether it is worthwhile to provide ulipristal acetate to minors free of charge. METHODS: The cost-effectiveness of two emergency contraceptive methods was compared based on a decision-analytical model. Pregnancy rates, outcomes of unintended pregnancies, and resource utilization were derived from the literature. Resources and their costs were considered until termination or a few days after delivery. Deterministic and probabilistic sensitivity analyses were performed. RESULTS: The cost of an unintended pregnancy in a French minor is estimated to be 1,630 € (range 1,330 € - 1,803 €). Almost 4 million € (3.1 € - 13.7 € million) in unintended pregnancy spending in 2010 could have been saved by the use of ulipristal acetate instead of levonorgestrel. The incremental cost of ulipristal acetate compared to levonorgestrel is 3.30 € per intake, or 418 € per pregnancy avoided (intake within 72 hours). In the intake within 24 hours subgroup, ulipristal acetate was found to be more efficacious at a lower cost compared to levonorgestrel. CONCLUSIONS: Ulipristal acetate dominates levonorgestrel when taken within 24 hours after unprotected intercourse, i.e., it is more effective at a lower cost. When taken within 72 hours, ulipristal acetate is a cost- effective alternative to levonorgestrel, given that the cost of avoiding an additional pregnancy with ulipristal acetate is less than the average cost of these pregnancies. In the light of these findings, it is worthwhile to provide free access to minors.