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Given the key role of the IL-23/Th17 axis in the pathogenesis of moderate-to-severe plaque psoriasis, several specific inhibitors of the p19 subunit of IL-23 have been approved to treat this chronic inflammatory disease. Clinical data indicate that guselkumab, one such selective IL-23 inhibitor, achieves greater clinical efficacy compared with ustekinumab, which inhibits both IL-12 and IL-23 via binding their shared p40 subunit. To understand mechanisms underlying the enhanced efficacy observed with the p19 subunit of IL-23–specific inhibition, we explored cellular and molecular changes in skin of psoriasis patients treated with ustekinumab or guselkumab and in ustekinumab inadequate responders (Investigator’s Global Assessment of psoriasis score ≥ 2) subsequently treated with guselkumab (ustekinumab→guselkumab). Skin biopsies were collected pretreatment and posttreatment to assess histologic changes and molecular responses in ustekinumab- and guselkumab-treated patients. Serum cytokines and skin transcriptomics from the subset of ustekinumab→guselkumab-treated patients were also analyzed to characterize differential treatment effects. Ustekinumab and guselkumab demonstrated differential effects on secretion of pathogenic Th17-related cytokines induced by IL-23 in in vitro assays, which suggest guselkumab is a more potent therapeutic agent. Consistent with these findings, guselkumab elicited a significantly greater reduction in cellular and molecular psoriasis-related disease indicators than ustekinumab. In ustekinumab→guselkumab patients, suppression of serum IL-17A and IL-17F levels and neutralization of molecular scar and psoriasis-related gene markers in skin were significantly greater compared with patients continuing ustekinumab. This comparative study demonstrates that guselkumab inhibits psoriasis-associated pathology, suppresses Th17-related serum cytokines, and normalizes the psoriasis skin gene expression profile more effectively than ustekinumab.

Background Atopic dermatitis (AD) is a heterogeneous, chronic inflammatory skin disease linked to skin microbiome dysbiosis with reduced bacterial diversity and elevated relative abundance of Staphylococcus aureus (S. aureus). Objectives We aimed to characterize the yet incompletely understood association between the skin microbiome and patients' demographic and clinical cofactors in relation to AD severity. Methods The skin microbiome in 48 adult moderate-to-severe AD patients was investigated using next-generation deep sequencing (16S rRNA gene, V1–V3 region) followed by denoising (DADA2) to obtain amplicon sequence variant (ASV) composition. Results In lesional skin, AD severity was associated with S. aureus relative abundance (rS = 0.53, p

Treatment of inflammatory skin diseases, including atopic dermatitis (AD) and psoriasis, is undergoing transformative changes, highlighting the need to develop experimental models of skin inflammation in humans to predict treatment responses.We topically or intradermally administered four common sensitizers (dust mite (DM), diphencyprone (DPCP), nickel (Ni), and purified protein derivative (PPD)) to the backs of 40 healthy patients and the skin hypersensitivity response was biopsied and evaluated using immunohistochemistry, RNA-seq, and RT-PCR.All agents induced strong increases in cellular infiltrates (T-cells and dendritic cells) as compared to untreated skin (p .05), with variable T helper polarization. Overall, DPCP induced the strongest immune responses across all pathways, including innate immunity (IL-1α, IL-8), Th1 (IFNγ, CXCL10), Th2 (IL-5, CCL11), and Th17 (CAMP/LL37) products, as well as the highest regulatory tone (FOXP3, IL-34, IL-37) (FDR 0.01). Nickel induced Th17 (IL-17A), Th1 (CXCL10) and Th2 (IL-4R) immune responses to a lesser extent than DPCP (p .05). PPD induced predominantly Th1 (IFNγ, CXCL10, STAT1) and Th17 inflammation (IL-17A) (p .05). DM induced modulation of Th2 (IL-13, CCL17, CCL18), Th22 (IL-22), and Th17/Th22 (S100A7/9/12) pathways (p .05). Barrier defects that characterize both AD and psoriasis were best modeled by DPCP and Ni, followed by PPD, including downregulation of terminal differentiation (FLG, FLG2, LOR, LCEs), tight junction (CLDN1/CLDN8), and lipid metabolism (FA2H, FABP7)-related markers.Our data imply that DPCP induced the strongest immune response across all pathways, and barrier defects characteristic of AD and psoriasis.

PDF file - 54K, Primers and probes used for Quantitative RT-PCR assay.

PDF file - 53K, Selected upregulated canonical Pathways in IL-32 treated MyLa cells.

PDF file - 316K, Selected upregulated and down regulated genes of IL-32 treated MyLa and HH cells.

Frontal fibrosing alopecia (FFA) is a progressive, scarring alopecia of the frontotemporal scalp that poses a substantial burden on quality of life. Large-scale global profiling of FFA is lacking, preventing the development of effective therapeutics.To characterize FFA compared to normal and alopecia areata using broad molecular profiling and to identify biomarkers linked to disease severity.This cross-sectional study assessed 33,118 genes in scalp using RNA sequencing and 350 proteins in serum using OLINK high-throughput proteomics. Disease biomarkers were also correlated with clinical severity and a fibrosis gene set.Genes differentially expressed in lesional FFA included markers related to Th1 (IFNγ/CXCL9/CXCL10), T-cell activation (CD2/CD3/CCL19/ICOS), fibrosis (CXCR3/FGF14/FGF22/VIM/FN1), T-regulatory (FOXP3/TGFB1/TGFB3), and Janus kinase/JAK (JAK3/STAT1/STAT4) (Fold changes [FCH]1.5, FDR.05 for all). Only one protein, ADM, was differentially expressed in FFA serum compared to normal (FCH1.3, FDR.05). Significant correlations were found between scalp biomarkers (IL-36RN/IL-25) and FFA severity, as well as between JAK/STAT and fibrosis gene-sets (r.6; P .05).This study was limited by a small sample size and predominantly female FFA patients.Our data characterize FFA as an inflammatory condition limited to scalp, involving Th1/JAK skewing, with associated fibrosis and elevated T-regulatory markers, suggesting the potential for disease reversibility with JAK/STAT inhibition.

Background Although hidradenitis suppurativa (HS) shares some transcriptomic and cellular infiltrate features with psoriasis, their skin proteome remains unknown. Objective To define and compare inflammatory protein biomarkers of HS and psoriasis skin. Methods We assessed 92 inflammatory biomarkers in HS (n = 13), psoriasis (n = 11), and control skin (n = 11) using Olink high-throughput proteomics. We also correlated HS skin and blood biomarkers using proteomics and RNA sequencing. Results We identified 57 differentially expressed proteins (DEPs) in lesional psoriasis and 64 DEPs in lesional HS skin, compared to healthy controls. Both HS and psoriasis lesional skin demonstrated a significant upregulation of T helper 1 and T helper 17 proteins. Healthy-appearing perilesional HS skin had 63 DEPs compared to healthy controls. Nonlesional HS and psoriasis skin had 24 and 7 DEPs, respectively, compared to healthy controls. Tumor necrosis factor and 8 other proteins were significantly correlated with clinical severity in perilesional HS skin (2 cm from a nodule). Limitations Inclusion of only moderate-to-severe patients and the cohort size. Conclusion HS has a greater inflammatory profile and is more diffusely distributed compared with psoriasis. Proteins correlated with disease severity are potential disease mediators. Perilesional skin is comparably inflamed to lesional skin, suggesting the need to treat beyond skin nodules.

Introduction: Clinical trials are a vital component of translational science, providing crucial information on the efficacy and safety of new interventions and forming the basis for regulatory approval and/or clinical adoption. At the same time, they are complex to design, conduct, monitor, and report successfully. Concerns over the last two decades about the quality of the design and the lack of completion and reporting of clinical trials, characterized as a lack of “informativeness,” highlighted by the experience during the COVID-19 pandemic, have led to several initiatives to address the serious shortcomings of the United States clinical research enterprise. Methods and Results: Against this background, we detail the policies, procedures, and programs that we have developed in The Rockefeller University Center for Clinical and Translational Science (CCTS), supported by a Clinical and Translational Science Award (CTSA) program grant since 2006, to support the development, conduct, and reporting of informative clinical studies. Conclusions: We have focused on building a data-driven infrastructure to both assist individual investigators and bring translational science to each element of the clinical investigation process, with the goal of both generating new knowledge and accelerating the uptake of that knowledge into practice.

Diphencyprone (DPCP) is a hapten that causes a delayed-type hypersensitivity reaction when applied topically. It has clinical uses in the treatment of various conditions such as melanoma metastases, warts, and alopecia areata, but the mechanisms are currently not well understood in humans. To further characterize the immunologic effects of DPCP, the authors performed a proteomic analysis of normal skin of eight healthy volunteers following a single application of DPCP and compared them with placebo-treated skin from the same volunteers. A total of 96 proteins were examined using the Olink immuno-oncology panel at 3 days (peak response), 14 days (partially resolved response), and 120 days (completely resolved response). Our analysis revealed significant upregulation of markers of immune cell activation (interleukin [IL] 8), vascular and tissue remodeling (matrix metallopeptidase 12 [MMP12], nitric oxide synthase 3 [NOS3]), antineoplastic markers (granzyme B [GZMB]), and the Th1 axis (interferon gamma [IFNG], chemokine (C-X-C motif) ligand [CXCL] 9, CXCL10, CXCL11) at days 3 and 14 compared with placebo (p 0.05). In addition, several negative regulators of immune function such as programmed cell death 1 (PD1), programmed cell death ligand 1 (PDL1) (p 0.001), and lymphocyte activation gene 3 (LAG3) (p 0.05) were significantly upregulated at days 3 and 14. This induction of negative regulators may explain the seemingly paradoxical therapeutic benefits of DPCP in autoimmune conditions such as alopecia areata. The current analysis also indicated IL-4 upregulation only at day 3, followed by IL-12 upregulation only at day 14, suggesting a transient Th2 response followed by Th1 polarization. Overall, these data suggest a complex and evolving immunological delayed-type hypersensitivity response to a single application of DPCP over time. Future proteomic studies of samples from patients with melanoma metastases, warts, and alopecia areata treated long term with DPCP are needed to further evaluate its pharmacologic mechanisms.

BackgroundPsoriasis is a chronic, systemic inflammatory condition, primarily affecting skin and joints. It is an immune-mediated disease driven by IL-17-producing T cells in the skin. However, epidermal cells such as keratinocytes are now also recognized as essential contributors to pathogenesis. We hypothesized that, under immunosuppressive treatment, skin inflammation alters the effect of genetic variants on gene expression in key pathways and cell types within the skin tissue. Understanding dermal and epidermal cell dynamics in psoriasis may help identify novel therapeutic targets.MethodsDuring the “Psoriasis Treatment with Abatacept and Ustekinumab: A Study of Efficacy” (PAUSE) trial, we obtained longitudinal lesional and non-lesional skin biopsies from participants at baseline, during treatment, and after withdrawal of study medication. Using whole-tissue genome-wide gene expression and genotype data, we mapped expression quantitative trait loci (eQTLs). Using transcriptional data, we defined local skin inflammation status using Skin Psoriatic Inflammation Transcriptional Score (SPITS) comparing lesional and non-lesional baseline skin biopsies. We identified interactions between eQTLs and SPITS status, as well as other features. We then examined the resulting eQTL genes (eGenes) and eQTL SNPs (eSNPs) to detect key pathways containing the eGenes whose regulation was altered by inflammation status.ResultsWe report 953 significant eQTLs at a Bonferroni p-value threshold (0.05/7,475,856). We assessed inflammation status in each biopsy based on SPITS and identified 116 eQTLs that are modified by changes in SPITS (FDR < 0.20); these eQTLs largely overlapped with those that were modified by an IL-17 pathway score, which is defined by genes induced in IL-17 stimulated keratinocytes (97/116=83.62%). In contrast, we detected few eQTL interactions with treatment (N = 0) and psoriasis area and severity index (PASI) score (N = 2 at FDR< 0.05; N = 5 at FDR < 0.20). Using psoriatic skin single-cell gene expression (84/116 found in the single cell dataset), we observed that most SPITS-interacting eGenes are skin cell-specific (62/84=73.81%) rather than immune cell-specific. We found the most significant SPITS interaction eQTL to be rs1491377616-LCE3C, and both the eSNP and eGene are associated with psoriasis genetic risk. Finally, we found SPITS-interacting eSNPs are enriched in NRF2 transcription factor binding motifs and may regulate four eGenes in the NRF2 pathway.ConclusionsThis is the first eQTL study of psoriatic skin in a clinical setting. The study highlights the potential to use biospecimens from clinical trials to discover in vivo eQTL interactions with therapeutically relevant environmental variables. We found that eQTLs are largely modified by local factors rather than systemic factors, such as medication usage or global psoriasis disease activity. We observed that the majority of eQTLs that are modulated by local skin inflammation in psoriasis are expressed in dermal and epidermal cell types and not immune cell types. Our results suggest that the rs1491377616 risk allele may be modulatingLCE3Cexpression in keratinocytes to cause disease. These results suggest that the landscape of changing gene regulation in dermal and epidermal cell types may be an important component of psoriasis, and the dermal and epidermal genes may be therapeutic targets.

Background Monoclonal antibodies targeting the interleukin (IL)-23p19 subunit are effective in the treatment of inflammatory bowel diseases (IBD), but have different molecular attributes that may translate to differences in clinical efficacy. Within this class, guselkumab (GUS) is a fully human IgG1 monoclonal antibody with a native Fc region, and risankizumab (RIS) is a humanised IgG1 antibody with a mutated Fc region. Binding of these therapeutic antibodies to Fcγ receptor (FcγR) I, or CD64, is of particular interest, as CD64+ IL-23-producing myeloid cells are increased in the inflamed colon in IBD and correlated with endoscopic disease severity (Chapuy L, et al. Mucosal Immunol. 2019; Chapuy et al. J Crohns Colitis. 2020). Here, we compared functional characteristics of the antigen-binding and Fc regions of GUS and RIS. Methods IL-23 binding affinity of GUS and RIS was compared in vitro using KinExA and surface plasmon resonance. In vitro cellular potency of GUS and RIS was measured by impact on IL-23-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation in human peripheral blood mononuclear cells. Binding of GUS and RIS to FcγRs was assessed in cells transfected with individual FcγRs. In primary human “inflammatory” monocytes differentiated with granulocyte-macrophage colony-stimulating factor and interferon-γ (IFN-γ) followed by toll-like receptor stimulation to induce IL-23 production, binding of GUS and RIS to CD64 and capture of endogenously secreted IL-23 was assessed using flow cytometry. The potential impact of GUS binding to CD64 was assessed in IFN-γ primed monocytes using a human 41-plex cytokine bead assay. Results GUS and RIS displayed comparable picomolar binding affinity for IL-23 and equivalent high potency for inhibition of IL-23-induced STAT3 phosphorylation. GUS showed strongest binding to CD64 compared with other FcγRs, whereas RIS had negligible binding to any FcγR. GUS, but not RIS, showed dose-dependent Fc-mediated binding to CD64 in primary human “inflammatory” monocytes. Moreover, CD64-bound GUS was able to simultaneously capture IL-23 endogenously secreted from the same cells (Figure). GUS binding to CD64 on monocytes did not induce cytokine production. Conclusion GUS, but not RIS, can simultaneously bind CD64+ myeloid cells via its Fc region and neutralise IL-23 with high affinity and potency. Our in vitro data suggest a mechanistic benefit through enhanced localization of GUS within the inflamed colon, where CD64+ IL-23-producing myeloid cells are increased, and GUS can potently neutralise IL-23 at its source of production. These findings may contribute to differences in therapeutic profiles between antibodies.

The mechanisms driving alopecia areata (AA) are still unclear, hindering development of targeted therapeutics. Specific Th2 targeting with dupilumab in AA provides a unique opportunity to dissect its pathogenesis and explore the role of Th2 pathway.We evaluated changes in scalp biomarkers in AA patients (with and without concomitant atopy) randomized to weekly dupilumab or placebo for 24 weeks, followed by open-label dupilumab for 24 weeks. Changes in biomarker levels were measured at weeks 12, 24, and 48 and were also correlated with clinical hair regrowth.At week 24, preceding clinical hair regrowth outcomes, only dupilumab-treated patients presented significant suppression of cellular infiltrates, and multiple Th2-related, markers (CCL13/MCP-4, CCL18/PARC, CCL26/eotaxin-3, CCL24/Eotaxin-2), coupled with significant upregulation in the hair keratins. Th1-related suppression was evident later (week 48) when all patients received open-label dupilumab. Results were more pronounced in atopic AA patients, that showed 48% and 97% improvements in the lesional AA scalp profile at weeks 24 and 48, respectively, while 2% worsening was seen in the placebo arm at week 24. Moreover, placebo-treated patients presented 54% worsening in hair keratins when compared with baseline at week 24. At week 24, increases in hair keratins showed significant correlations only with decreases in Th2-related markers.Scalp biomarkers provide evidence of dupilumab efficacy in AA, detected even prior to clinical response, with exclusive correlations between early suppression of Th2 markers and increased hair keratins. These findings strengthen previous reports suggesting a possible role for Th2 cytokines as AA drivers.

Generalized pustular psoriasis (GPP) is a rare, severe, clinically heterogeneous disease characterized by flares of widespread, noninfectious, macroscopically visible pustules that occur with or without systemic inflammation, and are associated with significant morbidity and mortality. Historically, GPP has been classified as a variant of psoriasis vulgaris (PV, or plaque psoriasis); however, accumulating evidence indicates that these are distinct conditions, requiring different treatment approaches.In this perspective article we review evidence that supports the classification of GPP as distinct from PV.The histopathologic and clinical appearance of GPP is distinct from that of PV and fundamental differences exist between the two conditions in terms of genetic causes and expression-related mechanisms of disease development. GPP results from dysregulation of the innate immune system, with disruption of the interleukin (IL)-36 inflammatory pathway, induction of inflammatory keratinocyte responses, and recruitment of neutrophils. PV is driven by the adaptive immune system, with a key role played by IL-17. Considering GPP as a separate disease will enable greater focus on its specific pathogenesis and the needs of patients. Many treatments for PV have insufficient efficacy in GPP and a therapeutic approach developed specifically for GPP might lead to better patient outcomes.Generalized pustular psoriasis (GPP) is a rare disease. During episodes of worsening disease, the immune system attacks the skin. This causes large areas of skin to become red and painful, pus-filled blisters suddenly form. Some people with GPP have a history of another, more common, skin condition called psoriasis vulgaris (PV). People with PV develop patches of scaly, itchy skin. In the past, GPP was classed as a type of PV and treated with the same medicines. However, these medicines do not work well in GPP. Researchers now understand more about what causes GPP and how it differs from PV. GPP can cause medical problems throughout the body, leading to life-threatening complications. This means that people with GPP often need urgent medical treatment in hospitals. People with PV are mostly treated outside of hospitals. Any other medical problems are not usually due to PV itself. Researchers have found several genes that are altered in people with GPP and PV, and they differ between the two diseases. For example, changes in a gene called

Summary Background Although alopecia areata (AA) greatly impacts patients’ quality of life (QoL), there is no adequate validation of AA-targeted QoL surveys in clinical trials, hindering sufficient representation of patient-reported outcomes. Objectives Better understanding of patient-reported outcomes may guide treatment goals and future clinical trials. Methods In a recent randomized controlled trial testing dupilumab in AA, patients were administered the Alopecia Areata Quality of Life Index (AA-QLI) and the Alopecia Areata Symptom Impact Scale (AASIS) surveys, specifically evaluating QoL in patients with AA. An in-depth analysis was performed to assess the utility of these questionnaires in this patient population, both at baseline and after treatment, and to determine a threshold for improved patient-reported outcomes. Results While AASIS correlated with baseline Severity of Alopecia Tool (SALT) scores and with therapeutic response, AA-QLI showed no correlation with AA severity before or after treatment. Itch strongly correlated with serum IgE levels across both surveys. Using various approaches to estimate a discriminative threshold for decreased impact of AA on QoL (by AASIS) following treatment, a SALT score of 20 points or less post-treatment was associated with improved patient-reported outcomes, including both AA-related symptoms and items within the daily activities/feelings domain such as ‘feeling sad’ and ‘feeling anxious or worry’. Conclusions AASIS is better than AA-QLI to assess patient-reported outcomes. SALT ≤ 20 following treatment should be considered as a threshold for meaningful therapeutic outcome and as a clinical endpoint in future clinical trials for AA. What is already known about this topic? Alopecia areata greatly compromises quality of life, and affected patients have increased prevalences of depression, anxiety and social phobia.Despite the significant negative impact of the disease on patients’ wellbeing, validation of targeted questionnaires in alopecia areata is lacking, and a therapeutic response threshold for improved patient-reported outcomes is unknown. What does this study add? This study investigated the utility of two different alopecia areata-targeted questionnaires – Alopecia Areata Quality of Life Index and Alopecia Areata Symptom Impact Scale (AASIS) – in a clinical trial setting.AASIS was found to correlate strongly with alopecia areata severity and clinical response. What are the clinical implications of this work? Patients with ≤ 20% scalp hair loss after treatment reported improvement in multiple quality-of-life items, suggesting this as a meaningful therapeutic outcome that may guide clinicians and improve the development of future clinical trials.

Psoriasis increases the risk of cardiovascular disease (CVD). Biomarkers for cardiovascular (CV) risk stratification in psoriasis are lacking, and the effects of psoriasis biologics on CV risk reduction remain unclear. The goal of this study was to identify biomarkers of CV risk in psoriasis blood that are reduced by ustekinumab. We quantified 276 inflammatory and CV-related serum proteins with Olink's multiplex assay in 10 psoriasis patients (vs. 18 healthy controls) and after 12 weeks of ustekinumab treatment. For each protein down-regulated after treatment, the literature was reviewed for studies assessing the protein's association with CVD. Data were collected from each study to calculate CV risk thresholds for each protein, which were compared with protein levels in psoriasis patients before and after treatment. Our results showed that 43 out of 276 proteins were down-regulated after treatment, 25 of which were initially up-regulated at baseline (vs. controls, all p-values ≤0.1). 8 down-regulated proteins were initially elevated above thresholds associated with enhanced CV risk in the literature (myeloperoxidase, C-X-C motif chemokine 10, E-selectin, interleukin-6, cystatin B, von Willebrand factor, tumor necrosis factor receptor 1 and N-terminal prohormone brain natriuretic peptide). Treatment lowered these proteins to below their risk thresholds, except for IL-6, which was lowered but remained at its risk threshold despite successful psoriasis skin treatment. In summary, 12 weeks of ustekinumab treatment reduced serum proteins present at levels associated with CV risk in psoriasis patients. Further studies can evaluate these proteins as potential ustekinumab-modulated biomarkers of CV risk in psoriasis and the impact of ustekinumab on CV risk reduction.

The frequency of salt marsh dieback events has increased over the last 25 years with unknown consequences to the resilience of the ecosystem to accelerated sea level rise (SLR). Salt marsh ecosystems impacted by sudden vegetation dieback events were previously thought to recover naturally within a few months to years. In this study, we provide evidence that approximately 14% of total marsh area has not revegetated 10-years after a dieback event in Charleston, SC. Dieback onset coincided with a severe drought in 2012, and a second dieback event occurred in 2016 after a historic flood influenced by Hurricane Joaquin in October of 2015, with unvegetated zones reaching nearly 30% of total marsh area in 2017. Most affected areas were associated with lower elevation zones in the interior of the marsh (midmarsh). During the 2013 dieback event, we estimate that unvegetated midmarsh area expanded by 300%. Grass planting was shown to be an effective restoration practice, with restored plants having greater aboveground biomass than relict sites after two years of transplanting. A positive restoration outcome indicated that the stressors that caused the initial dieback are no longer present. Despite that, many dieback areas have not recovered naturally even as they are located within the typical elevation range of a healthy vegetated marsh. A mechanistic modelling approach was used to assess the effects of vegetation dieback on salt marsh resilience to SLR. Predictions indicate that a highly productive restored marsh (2000 g m-2 y-1) would persist at a moderate SLR rate of 60 cm 100 y-1, whereas a non- restored mudflat would lose all of its elevation capital after 100 years. Thus, rapid restoration of marsh dieback is critical to avoid further degradation. Also, failure to incorporate the increasing frequency and intensity of extreme climatic events which trigger irreversible marsh diebacks underestimates salt marsh vulnerability to climate change. At an elevated SLR rate of 122 cm 100 y-1, most likely an extreme climate change scenario, even highly productive ecosystems augmented by sediment placement would not keep pace with SLR. Thus, climate change mitigation actions are also urgently needed to preserve present-day marsh ecosystems.

Background DNA methylation alterations have emerged as hallmarks of cancer and have been proposed as screening, prognostic, and predictive biomarkers. Traditional approaches for methylation analysis have relied on bisulfite conversion of DNA, which can damage DNA and is not suitable for targeted gene analysis in low-input samples. Here, we have adapted methyl-CpG-binding domain protein 2 (MBD2)-based DNA enrichment for use on a semi-automated exclusion-based sample preparation (ESP) platform for robust and scalable enrichment of methylated DNA from low-input samples, called SEEMLIS. Results We show that combining methylation-sensitive enzyme digestion with ESP-based MBD2 enrichment allows for single gene analysis with high sensitivity for GSTP1 in highly impure, heterogenous samples. We also show that ESP-based MBD2 enrichment coupled with targeted pre-amplification allows for analysis of multiple genes with sensitivities approaching the single cell level in pure samples for GSTP1 and RASSF1 and sensitivity down to 14 cells for these genes in highly impure samples. Finally, we demonstrate the potential clinical utility of SEEMLIS by successful detection of methylated gene signatures in circulating tumor cells (CTCs) from patients with prostate cancer with varying CTC number and sample purity. Conclusions SEEMLIS is a robust assay for targeted DNA methylation analysis in low-input samples, with flexibility at multiple steps. We demonstrate the feasibility of this assay to analyze DNA methylation in prostate cancer cells using CTCs from patients with prostate cancer as a real-world example of a low-input analyte of clinical importance. In summary, this novel assay provides a platform for determining methylation signatures in rare cell populations with broad implications for research as well as clinical applications.

LL37 is produced by skin injury and bacterial infection and plays an important role in the early stages of psoriasis. In particular, the intracellular receptors TLRs 3, 7, 8, and 9 are thought to be involved in the pathogenesis of psoriasis in conjunction with LL37, but the interaction between TLR7/8 and LL37 in keratinocytes remains unclear. This study aimed to clarify the relationship between LL37 and TLR7/8 in keratinocytes and their involvement in the pathogenetic pathways seen in psoriasis using cultured keratinocytes and psoriasis patient skin samples. TLR7/8 was induced by LL37 in keratinocytes. TLR8 but not TLR7 functionally induced many psoriasis-related molecules, whereas IL-17C was not altered by the blockade of TLR7/8. While co-stimulated of LL37 with self-RNA/DNA did not show any interaction, LL37 itself would promote psoriasis-related genes. IL-36 receptor antagonistic antibody suppressed IL-17C induced by LL37. In psoriatic epidermis, LL37, TLRs, IL-17C, and IL-36γ expressions were increased and co-expressed with each other. Thus, we concluded that LL37 activates TLR8 in keratinocytes and induces IL-17C via induction of IL-36γ. Regulation of TLR8 or LL37 in keratinocytes could be a potential therapeutic strategy for psoriatic inflammation.

The interaction between A-type interflavan bonds from cranberry proanthocyanidins (PAC) and surface virulence factors of extra-intestinal pathogenic Escherichia coli (ExPEC) was studied. Electrospun nanofibers (ESNF) were fabricated using PAC and polycaprolactone (PCL) solutions and their physical and chemical properties were characterized. The ability of PAC:PCL composite ESNF to interact with and entrap ExPEC strain 5011 (ExPEC-5011) was evaluated in vitro by plate culturing and when formulated as a biofilter and nanocoating. As a biofilter, the PAC:PCL ESNF exhibited a dose-dependent ability to entrap ExPEC-5011. Images from scanning electron and fluorescent microscopies revealed that ESNF sections with higher amounts of PAC led to higher bacterial entrapment. The effectiveness PAC:PCL ESNF to bind ExPEC when applied as a nanocoating was studied using ESNF-coated polyvinyl chloride intermittent catheter. Results indicate that ExPEC-5011 was entrapped well into the PAC:PCL ESNF coating on the catheter. Overall, our results suggest that incorporating the biomolecule PAC in ESNF is a potential means for applications requiring bacterial entrapment, such as biofunctionalization, biofiltration, and surface coating, among others.

Psoriasis vulgaris is a common inflammatory disease affecting 7.5 million adults just in the US. Previously, psoriasis immunopathogenesis has been viewed as the imbalance between CD4+ T-helper 17 (Th17) cells and regulatory T-cells (Tregs). However, current paradigms are rapidly evolving as new technologies to study immune cell subsets in the skin have been advanced. For example, recently minted single-cell RNA sequencing technology has provided the opportunity to compare highly differing transcriptomes of Type 17 T-cell (T17 cell) subsets depending on IL-17A vs. IL-17F expression. The expression of regulatory cytokines in T17 cell subsets provided evidence of T-cell plasticity between T17 cells and regulatory T-cells (Tregs) in humans. In addition to Tregs, other types of regulatory cells in the skin have been elucidated, including type 1 regulatory T-cells (Tr1 cells) and regulatory dendritic cells. More recently, investigators are attempting to apply single-cell technologies to clinical trials of biologics to test if monoclonal blockade of pathogenic T-cells will induce expansion of regulatory immune cell subsets involved in skin homeostasis.

Palmoplantar pustular psoriasis (PPPP) and non‒pustular palmoplantar psoriasis (NPPP) are localized, debilitating forms of psoriasis. The inflammatory circuits involved in PPPP and NPPP are not well-understood. To compare the cellular and immunological features that differentiate PPPP and NPPP, skin biopsies were collected from a total of 30 participants with PPPP, NPPP, and psoriasis vulgaris (PV) and from 10 healthy participants. A subset consented to a second biopsy after 3 additional weeks off medication. Histologic staining of lesional and nonlesional skin showed higher neutrophil counts in PPPP than in NPPP and PV and higher CD8

Pathogenic elements and potential treatment targets in hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is an inflammatory skin disease with dysregulation of the interleukin (IL)-17 axis. Recently, we reported the clinical benefit of brodalumab, a human anti-IL-17 receptor A (IL-17RA) monoclonal antibody, in moderate-to-severe HS.To characterize the molecular response to brodalumab in HS skin and serum, and to identify biomarkers of treatment response.Ten participants, who received brodalumab 210 mg /1·5 mL subcutaneously at weeks 0, 1, 2, 4 and every 2 weeks thereafter, were included in this molecular profiling study (NCT03960268). RNA sequencing and immunohistochemistry of nonlesional, perilesional and lesional HS skin biopsies, and Olink high-throughput proteomics of serum at baseline, weeks 4 and 12 were assessed.At week 12, brodalumab led to a decrease of overall inflammation, and improvement of psoriasis-, keratinocyte- and neutrophil-related pathways. Despite perilesional and lesional skin exhibiting no differentially expressed genes at baseline, treatment response was best assessed in perilesional skin. In serum, brodalumab treatment decreased pathways involved in neutrophil inflammation. Patients with higher baseline expression of neutrophil-associated lipocalin-2 (LCN2) in the skin or IL-17A in the serum demonstrated greater decreases of HS-related inflammatory cytokines as measured in skin biopsies at week 12.IL-17RA inhibition by brodalumab decreases several pathogenic inflammatory axes in HS. Perilesional skin provides a valid and robust assessment of treatment response. Expression of LCN2 in skin or IL-17A in serum may be used as biomarkers to stratify patients that may have a superior molecular response to brodalumab.

Background Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease presenting with diverse manifestations ranging from nodules and abscesses to draining tunnels. Whether the underlying inflammation from lesions extends to relatively healthy-appearing adjacent perilesional and distant nonlesional skin has not been systematically evaluated. Objective We sought to characterize lesional, perilesional, and nonlesional skin in patients with HS. Methods Skin biopsy samples were collected under ultrasound guidance from patients with active, untreated moderate-to-severe HS. Site-matched control biopsy samples from healthy volunteers were used for comparison. Results RNA sequencing demonstrated that HS skin clustered separately from healthy control skin, with perilesional and lesion skin clustering together and away from nonlesional skin. Immunohistochemistry analysis identified psoriasiform hyperplasia with keratin 16 positivity in both perilesional and lesional skin, with comparable levels of CD3+, CD11c+, and neutrophil elastase–positive cellular infiltration. There was a marked upregulation of IL-17 signaling in perilesional and lesional skin. HS samples clustered on the basis of expression of lipocalin-2 (LCN2), with samples characterized by high LCN2 expression in the skin exhibiting a differing transcriptomic profile with significantly higher overall inflammation than that of skin characterized by low LCN2 levels. Conclusions Perilesional HS skin has a transcriptomic and molecular profile comparable to that of lesional skin. HS can be grouped into 2 distinct subtypes based on molecular levels of LCN2 in the skin, with the LCN2-high subtype exhibiting an overall higher inflammatory burden and an upregulation of targetable cytokines. To our knowledge, this is the first study to characterize a unique HS subtype (and a potential endotype) that may guide future therapeutic targets.

Introduction La cellulite eosinophile (CE) est une affection cutanee rare caracterisee par des plaques urticariennes prurigineuses fixes et un infiltrat inflammatoire dermique polymorphe incluant de nombreux polynucleaires eosinophiles (PNEo). Les mecanismes physiopathologiques de la CE restent mal connus : le paradigme actuel supporte l’hypothese d’une hypersensibilite de type retarde mediee par l’interleukine (IL) 5. Les traitements standards reposent essentiellement sur des immunomodulateurs (ie corticosteroides, dapsone, ciclosporine) mais de nombreux patients restent resistants sans solution therapeutique precise. Le but de cette etude etait de mieux caracteriser les voies inflammatoires impliquees dans la CE. Materiel et methodes Nous avons analyse retrospectivement des echantillons de peau provenant de 12 patients atteints de CE et de 5 temoins sains, en utilisant la RT-qPCR et l’immunomarquage phospho-STAT (pSTAT). Nous avons ensuite explore la reponse clinique et moleculaire d’un patient refractaire traite avec le nouvel inhibiteur de JAK1/JAK2, le baricitinib. Resultats Nous avons observe une augmentation significative des transcriptions d’ARNm liees a des marqueurs specifiques de l’inflammation de type 2 (IL-13 et IL-3R, mais pas IL-5) et du recrutement des eosinophiles (CCL17, CCL18 et CCL26) dans la peau lesionnelle des patients atteints de CE comparativement aux temoins. Nous avons egalement observe que presque tous les echantillons de CE etaient fortement positifs pour pSTAT-5 et, a un moindre degre, pour pSTAT-1. En comparaison, la coloration de pSTAT-3 etait negative. Comme les pSTAT-1 et -5 sont classiquement associees a l’activation de JAK1/2, nous avons ensuite traite un patient EC refractaire (echec corticosteroides, dapsone, ciclosporine et lesions actives depuis plus de 3 ans) avec du baricitinib a 4 mg/jour. Apres 1 mois de traitement le patient etait totalement blanchi et sans recidive a ce jour. De facon concomitante, les marqueurs d’inflammation de type 2, notamment IL-13, IL-3, CCL17, CCL18 et CCL26 ont ete completement normalises dans la peau guerie apres le traitement par baricitinib. Discussion Nos resultats suggerent que la CE est liee a une inflammation cutanee de type 2 avec une activation essentiellement de la voie pSTAT5 et une elevation importante des IL13 et IL3, mais pas de l’IL5. Le retrocontrole negatif de l’IL3, classiquement produite par les PNEo, sur la production d’IL5 et de son recepteur, pourrait expliquer ce resultat. Enfin, ces resultats suggerent l’efficacite des traitements ciblant specifiquement l’inflammation de type 2 dans cette pathologie et ces traitements pourraient presenter un interet majeur dans formes refractaires.