Rapid Assay Diagnostic for Acute Stroke Recognition (RADAR): study protocol for a diagnostic accuracy study.

INTRODUCTION: Large-vessel occlusion (LVO) stroke is effectively treated by time-critical thrombectomy, a highly specialised procedure only available in a limited number of centres. Many patients with suspected stroke are admitted to their nearest hospital and require transfer to access treatment, with resulting delays. This study is evaluating the accuracy of a new rapid portable test for LVO stroke which could be used in the future to select patients for direct admission to a thrombectomy centre. METHODS AND ANALYSIS: Rapid Assay Diagnostic for Acute Stroke Recognition (RADAR) is a prospective observational cohort study taking place in stroke units in England. Participants are adults with a new suspected stroke with at least one face, arm or speech (FAST) symptom(s) and known onset within 6 hours or last known to be well 6-24 hours ago. The index test ('LVOne test' (Upfront Diagnostics)), consists of two portable lateral flow assays which use fingerprick capillary blood to detect d-dimer and glial fibrillary acidic protein concentrations. Reference standards comprise independently adjudicated standard CT/MRI brain±CT/MR angiography with senior clinician opinion to establish: ischaemic stroke±LVO; intracerebral haemorrhage; transient ischaemic attack; stroke mimic. Analyses will report sensitivity, specificity and negative and positive predictive values for identification of LVO stroke. Powered using a primary analysis population (≥2 FAST symptoms and known onset within 6 hours), 276 participants will detect a test specificity of 92%. The broader total study population which allows evaluation of the test for milder symptoms and unknown onset times is estimated to be 552 participants. ETHICS AND DISSEMINATION: Ethical (North East-Newcastle & North Tyneside 2 Research Ethics Committee (reference: 23/NE/0043), Health Research Authority and participating National Health Service Trust approvals are granted. Consent is required for enrolment. Dissemination of results will include presentations at conferences, publication in journals and plain English summaries. TRIAL REGISTRATION NUMBER: ISRCTN12414986.

Stroke survivor views on ambulance redirection as a strategy to increase access to thrombectomy in England.

Introduction: Intravenous thrombolysis and mechanical thrombectomy are effective time-sensitive treatments for selected cases of acute ischaemic stroke. While thrombolysis is widely available, thrombectomy can only be provided at facilities with the necessary equipment and interventionists. Suitable patients admitted to other hospitals require secondary transfer, causing delays to treatment. Pre-hospital ambulance redirection to thrombectomy facilities may improve access but treatment eligibility cannot be confirmed pre-hospital. Some redirected patients would travel further and be displaced without receiving thrombectomy. This study aimed to elicit stroke survivor and carer/relative views about the possible consequences of introducing a conceptual, idealised ambulance redirection pathway. Methods: Focus groups were undertaken using a topic guide describing four hypothetical ambulance redirection scenarios and their possible consequences: earlier treatment with thrombectomy; delayed diagnosis of non-stroke 'mimic' conditions; delayed thrombolysis treatment; and delayed diagnosis of haemorrhagic stroke. Meetings were audio recorded, transcribed verbatim and data analysed thematically using emergent coding. Results: Fifteen stroke survivors and carers/relatives participated in three focus groups. There was wide acceptance of possible low-risk consequences of ambulance redirection, including extended travel time, being further from home and experiencing longer hospital stays. Participants were more uncertain about higher-risk consequences, including delays in diagnosis/treatment for patients unsuitable for thrombectomy, but remained positive about ambulance redirection overall. Participants rationalised acceptance of higher-risk consequences by recognising that redirected patients would still access appropriate treatment, even if delayed. In addition, acceptance of ambulance redirection would be increased if there were robust clinical evidence showing net benefit over secondary transfer pathways. Conclusions: Participant views were generally supportive of ambulance redirection to facilitate access to thrombectomy. Further research is needed to demonstrate overall benefit in an NHS context.

Healthcare professional views about a prehospital redirection pathway for stroke thrombectomy: a multiphase deductive qualitative study.

BACKGROUND: Mechanical thrombectomy for stroke is highly effective but time-critical. Delays are common because many patients require transfer between local hospitals and regional centres. A two-stage prehospital redirection pathway consisting of a simple ambulance screen followed by regional centre assessment to select patients for direct admission could optimise access. However, implementation might be challenged by the limited number of thrombectomy providers, a lack of prehospital diagnostic tests for selecting patients and whether finite resources can accommodate longer ambulance journeys plus greater central admissions. We undertook a three-phase, multiregional, qualitative study to obtain health professional views on the acceptability and feasibility of a new pathway. METHODS: Online focus groups/semistructured interviews were undertaken designed to capture important contextual influences. We purposively sampled NHS staff in four regions of England. Anonymised interview transcripts underwent deductive thematic analysis guided by the NASSS (Non-adoption, Abandonment and Challenges to Scale-up, Spread and Sustainability, Implementation) Implementation Science framework. RESULTS: Twenty-eight staff participated in 4 focus groups, 2 group interviews and 18 individual interviews across 4 Ambulance Trusts, 5 Hospital Trusts and 3 Integrated Stroke Delivery Networks (ISDNs). Five deductive themes were identified: (1) (suspected) stroke as a condition, (2) the pathway change, (3) the value participants placed on the proposed pathway, (4) the possible impact on NHS organisations/adopter systems and (5) the wider healthcare context. Participants perceived suspected stroke as a complex scenario. Most viewed the proposed new thrombectomy pathway as beneficial but potentially challenging to implement. Organisational concerns included staff shortages, increased workflow and bed capacity. Participants also reported wider socioeconomic issues impacting on their services contributing to concerns around the future implementation. CONCLUSIONS: Positive views from health professionals were expressed about the concept of a proposed pathway while raising key content and implementation challenges and useful 'real-world' issues for consideration.

A human neural crest model reveals the developmental impact of neuroblastoma-associated chromosomal aberrations.

Early childhood tumours arise from transformed embryonic cells, which often carry large copy number alterations (CNA). However, it remains unclear how CNAs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analysis to assess the effects of chromosome 17q/1q gains, which are prevalent in the embryonal tumour neuroblastoma (NB). We show that CNAs impair the specification of trunk neural crest (NC) cells and their sympathoadrenal derivatives, the putative cells-of-origin of NB. This effect is exacerbated upon overexpression of MYCN, whose amplification co-occurs with CNAs in NB. Moreover, CNAs potentiate the pro-tumourigenic effects of MYCN and mutant NC cells resemble NB cells in tumours. These changes correlate with a stepwise aberration of developmental transcription factor networks. Together, our results sketch a mechanistic framework for the CNA-driven initiation of embryonal tumours.

Kinase-inactivated CDK6 preserves the long-term functionality of adult hematopoietic stem cells.

ABSTRACT: Hematopoietic stem cells (HSCs) are characterized by the ability to self-renew and to replenish the hematopoietic system. The cell-cycle kinase cyclin-dependent kinase 6 (CDK6) regulates transcription, whereby it has both kinase-dependent and kinase-independent functions. Herein, we describe the complex role of CDK6, balancing quiescence, proliferation, self-renewal, and differentiation in activated HSCs. Mouse HSCs expressing kinase-inactivated CDK6 show enhanced long-term repopulation and homing, whereas HSCs lacking CDK6 have impaired functionality. The transcriptomes of basal and serially transplanted HSCs expressing kinase-inactivated CDK6 exhibit an expression pattern dominated by HSC quiescence and self-renewal, supporting a concept, in which myc-associated zinc finger protein (MAZ) and nuclear transcription factor Y subunit alpha (NFY-A) are critical CDK6 interactors. Pharmacologic kinase inhibition with a clinically used CDK4/6 inhibitor in murine and human HSCs validated our findings and resulted in increased repopulation capability and enhanced stemness. Our findings highlight a kinase-independent role of CDK6 in long-term HSC functionality. CDK6 kinase inhibition represents a possible strategy to improve HSC fitness.

A qualitative exploration of ambulance clinician behaviour and decision making to identify factors influencing on-scene times for suspected stroke patients in North East England.

Aims/objectives: Ambulance clinician assessment of suspected stroke patients aims to provide rapid access to specialist care, however regional and national data show increasing pre-hospital times. This study explored paramedic views about factors contributing to on-scene time (OST) for suspected stroke patients, with a view to identifying opportunities for future interventions, to reduce OST. Methods: Views of paramedics from one regional service on factors influencing OST were explored using a qualitative approach. Semi-structured interviews with volunteers were recorded, transcribed and analysed using thematic analysis. Results: Interviews were conducted with 13 paramedics between August and November 2021. Five interlinked themes were identified and described a range of factors influencing OST: 'Initial assessment and sources of information' describes how clinicians make assessments based on initial presentation, influenced by pre-arrival information from ambulance control and family members / bystanders at the scene, and how this influences OST.'Suitability for treatment and interventions' describes how paramedics consider actions such as the face, arms, speech test, cannulation, electrocardiograms and neurological assessments while recognising that pre-hospital interventions for suspected stroke are limited.'The environment' describes the influence of incident setting on OST, including the overall process needed to transport the patient to appropriate care.'Hospital interactions' describes how interactions with hospital staff influenced paramedic actions and OST.'Changing practice' describes the influence of experience and interaction with hospital staff leading to changes in paramedic practice over time. Conclusion: This study provides insight into how UK paramedics spend time on scene with stroke patients. Multiple factors influencing OST were identified which signpost opportunities for interventions designed to reduce OST. Standardising on-scene assessments for stroke patients, refining communication processes between ambulance services and hospital stroke services and increasing availability of stroke continuing professional development for paramedics were all identified as potential targets for improving OST.

Assessing mid-career female physician burnout in the military health system: finding joy in practice after the COVID-19 pandemic.

BACKGROUND: Rates of physician burnout increased during the COVID-19 pandemic and are expected to continue to rise. Mid-career physicians, female physicians, and military physicians have all been identified as potentially vulnerable populations to experience burnout. We examine factors associated with physician burnout among this intersectional group through a qualitative key informant interview study. METHODS: We developed a semi-structured interview guide using the Institute for Healthcare Improvement's Improving Joy in Work Framework and recruited military, mid-career female physicians who worked in the Military Health System(MHS) during the COVID-19 pandemic, (March 2020 -December 2021). Notes were collated and deductive thematic analysis was conducted. RESULTS: We interviewed a total of 22 mid-career female physician participants. Participants were between 30 and 44 years of age and 7 were mothers during the pandemic. Most were White and served in the Army. All participants discussed the importance of building rapport and having a good relationship with coworkers. All participants also described their discontentment with the new MHS GENESIS electronic health record system. An emerging theme was military pride as most participants were proud to serve in and support the military population. Additionally, participants discussed the negative impact from not feeling supported and not feeling heard by leadership. CONCLUSIONS: Much like providers in other health systems during the pandemic, MHS physicians experienced burnout. This study allowed us to gather key insights to improve policies for active duty service mid-career female military physicians. Provider inclusion, autonomy, and work culture play critical roles in future systems improvement and workforce retention.

Coordinated ARP2/3 and glycolytic activities regulate the morphological and functional fitness of human CD8+ T cells.

Actin cytoskeleton remodeling sustains the ability of cytotoxic T cells to search for target cells and eliminate them. We here investigated the relationship between energetic status, actin remodeling, and functional fitness in human CD8+ effector T cells. Cell spreading during migration or immunological synapse assembly mirrored cytotoxic activity. Morphological and functional fitness were boosted by interleukin-2 (IL-2), which also stimulated the transcription of glycolytic enzymes, actin isoforms, and actin-related protein (ARP)2/3 complex subunits. This molecular program scaled with F-actin content and cell spreading. Inhibiting glycolysis impaired F-actin remodeling at the lamellipodium, chemokine-driven motility, and adhesion, while mitochondrial oxidative phosphorylation blockade impacted cell elongation during confined migration. The severe morphological and functional defects of ARPC1B-deficient T cells were only partially corrected by IL-2, emphasizing ARP2/3-mediated actin polymerization as a crucial energy state integrator. The study therefore underscores the tight coordination between metabolic and actin remodeling programs to sustain the cytotoxic activity of CD8+ T cells.

Comparative transcriptomics coupled to developmental grading via transgenic zebrafish reporter strains identifies conserved features in neutrophil maturation.

Neutrophils are evolutionarily conserved innate immune cells playing pivotal roles in host defense. Zebrafish models have contributed substantially to our understanding of neutrophil functions but similarities to human neutrophil maturation have not been systematically characterized, which limits their applicability to studying human disease. Here we show, by generating and analysing transgenic zebrafish strains representing distinct neutrophil differentiation stages, a high-resolution transcriptional profile of neutrophil maturation. We link gene expression at each stage to characteristic transcription factors, including C/ebp-ß, which is important for late neutrophil maturation. Cross-species comparison of zebrafish, mouse, and human samples confirms high molecular similarity of immature stages and discriminates zebrafish-specific from pan-species gene signatures. Applying the pan-species neutrophil maturation signature to RNA-sequencing data from human neuroblastoma patients reveals association between metastatic tumor cell infiltration in the bone marrow and an overall increase in mature neutrophils. Our detailed neutrophil maturation atlas thus provides a valuable resource for studying neutrophil function at different stages across species in health and disease.

Exploring the heterogeneous transcriptional response of the CNS to systemic LPS and Poly(I:C).

Peripheral contact to pathogen-associated molecular patterns (PAMPs) evokes a systemic innate immune response which is rapidly relayed to the central nervous system (CNS). The remarkable cellular heterogeneity of the CNS poses a significant challenge to the study of cell type and stimulus dependent responses of neural cells during acute inflammation. Here we utilized single nuclei RNA sequencing (snRNAseq), serum proteome profiling and primary cell culture methods to systematically compare the acute response of the mammalian brain to the bacterial PAMP lipopolysaccharide (LPS) and the viral PAMP polyinosinic:polycytidylic acid (Poly(I:C)), at single cell resolution. Our study unveiled convergent transcriptional cytokine and cellular stress responses in brain vascular and ependymal cells and a downregulation of several key mediators of directed blood brain barrier (BBB) transport. In contrast the neuronal response to PAMPs was limited in acute neuroinflammation. Moreover, our study highlighted the dominant role of IFN signalling upon Poly(I:C) challenge, particularly in cells of the oligodendrocyte lineage. Collectively our study unveils heterogeneous, shared and distinct cell type and stimulus dependent acute responses of the CNS to bacterial and viral PAMP challenges. Our findings highlight inflammation induced dysregulations of BBB-transporter gene expression, suggesting potential translational implications on drug pharmacokinetics variability during acute neuroinflammation. The pronounced dependency of oligodendrocytes on IFN stimulation during viral PAMP challenges, emphasizes their limited molecular viral response repertoire.

Non-IgE-reactive allergen peptides deteriorate the skin barrier in house dust mite-sensitized atopic dermatitis patients.

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by type 2 cytokine-driven skin inflammation and epithelial barrier dysfunction. The latter is believed to allow the increased penetration of chemicals, toxins, and allergens into the skin. House dust mite allergens, particularly Der p 2, are important triggers in sensitized individuals with AD; the precise actions of these allergens in epithelial biology remain, however, incompletely understood. In this study, we compared the effects of the protein allergen Der p 2 and a mix of non-IgE-reactive Der p 2 peptides on skin cells using patch tests in AD patients and healthy participants. We then analyzed mRNA expression profiles of keratinocytes by single-cell RNA-sequencing. We report that existing barrier deficiencies in the non-lesional skin of AD patients allow deep penetration of Der p 2 and its peptides, leading to local microinflammation. Der p 2 protein specifically upregulated genes involved in the innate immune system, stress, and danger signals in suprabasal KC. Der p 2 peptides further downregulated skin barrier genes, in particular the expression of genes involved in cell-matrix and cell-cell adhesion. Peptides also induced genes involved in hyperproliferation and caused disturbances in keratinocyte differentiation. Furthermore, inflammasome-relevant genes and IL18 were overexpressed, while KRT1 was downregulated. Our data suggest that Der p 2 peptides contribute to AD initiation and exacerbation by augmenting hallmark features of AD, such as skin inflammation, barrier disruption, and hyperplasia of keratinocytes.

Associations between AI-Assisted Tumor Amphiregulin and Epiregulin IHC and Outcomes from Anti-EGFR Therapy in the Routine Management of Metastatic Colorectal Cancer.

PURPOSE: High tumor production of the EGFR ligands, amphiregulin (AREG) and epiregulin (EREG), predicted benefit from anti-EGFR therapy for metastatic colorectal cancer (mCRC) in a retrospective analysis of clinical trial data. Here, AREG/EREG IHC was analyzed in a cohort of patients who received anti-EGFR therapy as part of routine care, including key clinical contexts not investigated in the previous analysis. EXPERIMENTAL DESIGN: Patients who received panitumumab or cetuximab ± chemotherapy for treatment of RAS wild-type mCRC at eight UK cancer centers were eligible. Archival formalin-fixed paraffin-embedded tumor tissue was analyzed for AREG and EREG IHC in six regional laboratories using previously developed artificial intelligence technologies. Primary endpoints were progression-free survival (PFS) and overall survival (OS). RESULTS: A total of 494 of 541 patients (91.3%) had adequate tissue for analysis. A total of 45 were excluded after central extended RAS testing, leaving 449 patients in the primary analysis population. After adjustment for additional prognostic factors, high AREG/EREG expression (n = 360; 80.2%) was associated with significantly prolonged PFS [median: 8.5 vs. 4.4 months; HR, 0.73; 95% confidence interval (CI), 0.56-0.95; P = 0.02] and OS [median: 16.4 vs. 8.9 months; HR, 0.66 95% CI, 0.50-0.86; P = 0.002]. The significant OS benefit was maintained among patients with right primary tumor location (PTL), those receiving cetuximab or panitumumab, those with an oxaliplatin- or irinotecan-based chemotherapy backbone, and those with tumor tissue obtained by biopsy or surgical resection. CONCLUSIONS: High tumor AREG/EREG expression was associated with superior survival outcomes from anti-EGFR therapy in mCRC, including in right PTL disease. AREG/EREG IHC assessment could aid therapeutic decisions in routine practice. See related commentary by Randon and Pietrantonio, p. 4021.

Single-cell transcriptomics and epigenomics unravel the role of monocytes in neuroblastoma bone marrow metastasis.

Metastasis is the major cause of cancer-related deaths. Neuroblastoma (NB), a childhood tumor has been molecularly defined at the primary cancer site, however, the bone marrow (BM) as the metastatic niche of NB is poorly characterized. Here we perform single-cell transcriptomic and epigenomic profiling of BM aspirates from 11 subjects spanning three major NB subtypes and compare these to five age-matched and metastasis-free BM, followed by in-depth single cell analyses of tissue diversity and cell-cell interactions, as well as functional validation. We show that cellular plasticity of NB tumor cells is conserved upon metastasis and tumor cell type composition is NB subtype-dependent. NB cells signal to the BM microenvironment, rewiring via macrophage mgration inhibitory factor and midkine signaling specifically monocytes, which exhibit M1 and M2 features, are marked by activation of pro- and anti-inflammatory programs, and express tumor-promoting factors, reminiscent of tumor-associated macrophages. The interactions and pathways characterized in our study provide the basis for therapeutic approaches that target tumor-to-microenvironment interactions.

Cytokine-directed cellular cross-talk imprints synovial pathotypes in rheumatoid arthritis.

INTRODUCTION: Structural reorganisation of the synovium with expansion of fibroblast-like synoviocytes (FLS) and influx of immune cells is a hallmark of rheumatoid arthritis (RA). Activated FLS are increasingly recognised as a critical component driving synovial tissue remodelling by interacting with immune cells resulting in distinct synovial pathotypes of RA. METHODS: Automated high-content fluorescence microscopy of co-cultured cytokine-activated FLS and autologous peripheral CD4+ T cells from patients with RA was established to quantify cell-cell interactions. Phenotypic profiling of cytokine-treated FLS and co-cultured T cells was done by flow cytometry and RNA-Seq, which were integrated with publicly available transcriptomic data from patients with different histological synovial pathotypes. Computational prediction and knock-down experiments were performed in FLS to identify adhesion molecules for cell-cell interaction. RESULTS: Cytokine stimulation, especially with TNF-α, led to enhanced FLS-T cell interaction resulting in cell-cell contact-dependent activation, proliferation and differentiation of T cells. Signatures of cytokine-activated FLS were significantly enriched in RA synovial tissues defined as lymphoid-rich or leucocyte-rich pathotypes, with the most prominent effects for TNF-α. FLS cytokine signatures correlated with the number of infiltrating CD4+ T cells in synovial tissue of patients with RA. Ligand-receptor pair interaction analysis identified ICAM1 on FLS as an important mediator in TNF-mediated FLS-T cell interaction. Both, ICAM1 and its receptors were overexpressed in TNF-treated FLS and co-cultured T cells. Knock-down of ICAM1 in FLS resulted in reduced TNF-mediated FLS-T cell interaction. CONCLUSION: Our study highlights the role of cytokine-activated FLS in orchestrating inflammation-associated synovial pathotypes providing novel insights into disease mechanisms of RA.

Biallelic NFATC1 mutations cause an inborn error of immunity with impaired CD8+ T-cell function and perturbed glycolysis.

The nuclear factor of activated T cells (NFAT) family of transcription factors plays central roles in adaptive immunity in murine models; however, their contribution to human immune homeostasis remains poorly defined. In a multigenerational pedigree, we identified 3 patients who carry germ line biallelic missense variants in NFATC1, presenting with recurrent infections, hypogammaglobulinemia, and decreased antibody responses. The compound heterozygous NFATC1 variants identified in these patients caused decreased stability and reduced the binding of DNA and interacting proteins. We observed defects in early activation and proliferation of T and B cells from these patients, amenable to rescue upon genetic reconstitution. Stimulation induced early T-cell activation and proliferation responses were delayed but not lost, reaching that of healthy controls at day 7, indicative of an adaptive capacity of the cells. Assessment of the metabolic capacity of patient T cells revealed that NFATc1 dysfunction rendered T cells unable to engage in glycolysis after stimulation, although oxidative metabolic processes were intact. We hypothesized that NFATc1-mutant T cells could compensate for the energy deficit due to defective glycolysis by using enhanced lipid metabolism as an adaptation, leading to a delayed, but not lost, activation responses. Indeed, we observed increased 13C-labeled palmitate incorporation into citrate, indicating higher fatty acid oxidation, and we demonstrated that metformin and rosiglitazone improved patient T-cell effector functions. Collectively, enabled by our molecular dissection of the consequences of loss-of-function NFATC1 mutations and extending the role of NFATc1 in human immunity beyond receptor signaling, we provide evidence of metabolic plasticity in the context of impaired glycolysis observed in patient T cells, alleviating delayed effector responses.

What factors contribute towards ambulance on-scene times for suspected stroke patients? An observational study.

INTRODUCTION: Pre-hospital stroke care focusses on rapid access to specialist stroke units, but UK ambulance data shows increasing pre-hospital times. This study aimed to describe factors contributing towards ambulance on-scene times (OST) for suspected stroke patients and identify targets for a future intervention. PATIENTS AND METHODS: Ambulance clinicians in North East Ambulance Service were asked to complete a survey after transporting any suspected stroke patients to describe the patient encounter, interventions and timings. Completed surveys were linked with electronic patient care records. Potentially modifiable factors were identified by the study team. Poisson regression analysis quantified the association of selected potentially modifiable factors with OST. RESULTS: About 2037 suspected stroke patients were conveyed between July and December 2021, resulting in 581 fully completed surveys by 359 different clinicians. The median age of patients was 75 years (interquartile range (IQR) 66-83) and 52% of patients were male. Median OST was 33 min (IQR 26-41). Three potentially modifiable factors were identified as contributors to extended OST. Performing additional advanced neurological assessments added 10% to OST (34 vs 31 min, p = 0.008); intravenous cannulation added 13% (35 vs 31 min, p = <0.001) and ECGs added 22% (35 vs 28 min, p = <0.001). CONCLUSIONS: This study identified three potentially modifiable factors that increased pre-hospital OST with suspected stroke patients. This type of data can be used to target interventions at behaviours that extend pre-hospital OST but which have questionable patient benefit. This approach will be evaluated in a follow up study in the North East of England.

Single-cell RNA sequencing defines disease-specific differences between chronic nodular prurigo and atopic dermatitis.

BACKGROUND: Chronic nodular prurigo (CNPG) is an inflammatory skin disease that is maintained by a chronic itch-scratch cycle likely rooted in neuroimmunological dysregulation. This condition may be associated with atopy in some patients, and there are now promising therapeutic results from blocking type 2 cytokines such as IL-4, IL-13, and IL-31. OBJECTIVES: This study aimed to improve the understanding of pathomechanisms underlying CNPG as well as molecular relationships between CNPG and atopic dermatitis (AD). METHODS: We profiled skin lesions from patients with CNPG in comparison with AD and healthy control individuals using single-cell RNA sequencing combined with T-cell receptor sequencing. RESULTS: We found type 2 immune skewing in both CNPG and AD, as evidenced by CD4+ helper T cells expressing IL13. However, only AD harbored an additional, oligoclonally expanded CD8A+IL9R+IL13+ cytotoxic T-cell population, and immune activation pathways were highly upregulated in AD, but less so in CNPG. Conversely, CNPG showed signatures of extracellular matrix organization, collagen synthesis, and fibrosis, including a unique population of CXCL14-IL24+ secretory papillary fibroblasts. Besides known itch mediators such as IL31 and oncostatin M, we also detected increased levels of neuromedin B in fibroblasts of CNPG lesions compared with AD and HC, with neuromedin B receptors detectable on some nerve endings. CONCLUSIONS: These data show that CNPG does not harbor the strong disease-specific immune activation pathways that are typically found in AD but is rather characterized by upregulated stromal remodeling mechanisms that might have a direct impact on itch fibers.