Glycemic Changes and Weight Loss Precede Pancreatic Ductal Adenocarcinoma by up to 3 Years in a Diverse Population.

BACKGROUND & AIMS: Elevations in fasting blood glucose are observed prior to the development of pancreatic ductal adenocarcinoma (PDAC). Our aim was to describe glycemic and weight changes that occur prior to PDAC diagnosis in a diverse population. METHODS: We conducted a case-control study comparing patients with PDAC with matched controls between January 2011 and November 2019 at a tertiary care institution. Normally distributed variables were compared using t tests, and the Wilcoxon rank sum test was used for non-normally distributed variables; logistic regression was used to estimate odds of PDAC based on changes over time in hemoglobin A1c (HbA1c) and body mass index (BMI), controlling for appropriate confounders. RESULTS: A total of 4626 patients met inclusion criteria: 1542 cases and 3084 controls; the median age was 69.3 years, and 2487 (53.8%) were male; 751 cases (48.7%) were non-Hispanic white. In the 3 years prior to diagnosis, HbA1c was higher in patients with PDAC compared with controls (P ≤ .02 for all); a similar trend was seen for glucose values. BMI was greater for patients with PDAC for all study periods, except 0 to 6 months prior to cancer diagnosis when BMI was lower (P < .01 for all). The change in BMI (ΔBMI) of cases at 1 year and 6 months before diagnosis was -0.59 and -1.21 when compared with -0.08 and 0.03 for controls (P < .01 for both). Multivariable logistic regression demonstrated that HbA1c slope (adjusted odds ratio, 1.33; 95% confidence interval, 1.01-1.76) and BMI slope (adjusted odds ratio, 0.75; 95% confidence interval, 0.65-0.87) were predictors of PDAC. CONCLUSION: Glycemic elevations and weight loss predate PDAC diagnosis. These metabolic changes may suggest an underlying PDAC.

Scalp and serum profiling of frontal fibrosing alopecia reveals scalp immune and fibrosis dysregulation with no systemic involvement.

BACKGROUND: 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. OBJECTIVE: To characterize FFA compared to normal and alopecia areata using broad molecular profiling and to identify biomarkers linked to disease severity. METHODS: 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. RESULTS: 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 (FCH>1.3, 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). LIMITATIONS: This study was limited by a small sample size and predominantly female FFA patients. CONCLUSION: 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.

High-dimensional analysis defines multicytokine T-cell subsets and supports a role for IL-21 in atopic dermatitis.

BACKGROUND: Flow cytometry is a well-accepted approach for immune profiling; however, its value is restricted by the limited number of markers that can be analyzed simultaneously. Mass cytometry/CyTOF offers broad-scale immune characterization integrating large number of parameters. While partial blood phenotyping was reported in atopic dermatitis (AD), patients' comprehensive profiling, critical for leveraging new targeted treatments, is not available. IL-21 may be involved in inflammatory skin diseases but its role in AD is not well established. METHODS: We studied T-cell polarization in the blood of 20 moderate-to-severe AD and 15 controls. Using CyTOF and an unsupervised analysis, we measured the frequencies and mean metal intensities of activated polar CD4+ /CD8+ T-cell subsets. Immunohistochemistry, immunofluorescence, and qRT-PCR were used to analyze skin samples. RESULTS: Examining 24 surface, intracellular markers, and transcription factors, we identified six CD4+ and five CD8+ T-cell metaclusters. A CD4+ skin-homing IL-13+ monocytokine and a novel IL-13+ IL-21+ multicytokine metaclusters were increased in AD vs. controls (p < .01). While IL-13 signature characterized both clusters, levels were significantly higher in the IL-21+ group. Both clusters correlated with AD severity (r = 0.49, p = .029). Manual gating corroborated these results and identified additional multicytokine subsets in AD. Immunohistochemistry and immunofluorescence, validated by mRNA expression, displayed significantly increasedIL-21 counts and colocalization with IL-13/IL-4R in AD skin. CONCLUSION: A multicytokine signature characterizes moderate-to-severe AD, possibly explaining partial therapeutic responses to one cytokine targeting, particularly in severe patients. Prominent IL-21 signature in blood and skin hints for a potential pathogenic role of IL-21 in AD.

An integrated scalp and blood biomarker approach suggests the systemic nature of alopecia areata.

BACKGROUND: Alopecia areata (AA) is characterized by immune dysregulation in both scalp and blood, but a large-scale approach establishing biomarkers of AA incorporating both scalp tissue and serum compartments is lacking. We aimed to characterize the transcriptomic signature of AA lesional and nonlesional scalp compared to healthy scalp and determine its relationship with the blood proteome in the same individuals, with comparative correlations to clinical AA disease severity. METHODS: We evaluated lesional and nonlesional scalp tissues and serum from patients with moderate-to-severe AA (n = 18) and healthy individuals (n = 8). We assessed 33,118 genes in AA scalp tissue using RNAseq transcriptomic evaluation and 340 inflammatory proteins in serum using OLINK high-throughput proteomics. Univariate and multivariate approaches were used to correlate disease biomarkers with Severity of Alopecia Tool (SALT). RESULTS: A total of 608 inflammatory genes were differentially expressed in lesional AA scalp (fold change/FCH>1.5, false discovery rate/FDR<0.05) including Th1 (IFNG/IL12B/CXCL11), Th2 (IL13/CCL18), and T-cell activation-related (ICOS) products. Th1/Th2-related markers were significantly correlated with AA clinical severity in lesional/nonlesional tissue, while keratins (KRT35/KRT83/KRT81) were significantly downregulated in lesional compared to healthy scalp (p < .05). Expression of cardiovascular/atherosclerosis-related markers (MMP9/CCL2/IL1RL1/IL33R/ST2/AGER) in lesional scalp correlated with their corresponding serum expression (p < .05). AA scalp demonstrated significantly greater biomarker dysregulation compared to blood. An integrated multivariate approach combining scalp and serum biomarkers improved correlations with disease severity/SALT. CONCLUSION: This study contributes a unique understanding of the phenotype of moderate-to-severe AA with an integrated scalp and serum biomarker model suggesting the systemic nature of the disease, advocating for the need for immune-based systemic treatment.

Vascular inflammation in moderate-to-severe atopic dermatitis is associated with enhanced Th2 response.

BACKGROUND: In atopic dermatitis (AD), some studies have shown an association with increased cardiovascular disease in certain populations. However, other investigations found modest or no association. Despite conflicting results, molecular profiling studies in both AD skin and blood have demonstrated upregulation of atherosclerosis and cardiovascular risk-related markers. However, the underlying mechanisms connecting AD to vascular inflammation/atherosclerosis are unknown. In this study, we aim to determine factors associated with vascular inflammation/atherosclerosis in AD patients. METHODS: We used 18-FDG PET-CT to characterize vascular inflammation in AD patients and healthy subjects. In parallel, we assessed their skin and blood immune profiles to determine AD-related immune biomarkers associated with vascular inflammation. We also assessed levels of circulating microparticles, which are known to be associated with increased cardiovascular risk. RESULTS: We found significant correlations between vascular inflammation and Th2-related products in skin and blood of AD patients as well as atherosclerosis-related markers that were modulated by dupilumab. Circulating levels of endothelial microparticles were significantly higher in severe AD patients and tended to correlate with vascular inflammation assessed by PET-CT. CONCLUSION: Vascular inflammation in AD is associated with enhanced Th2 response and clinical severity, which may explain cardiovascular comorbidities observed in select AD populations. Larger prospective studies are needed to further evaluate vascular inflammation and cardiovascular events and mortality in AD patients. Finally, as dupilumab treatment demonstrated significant modulation of atherosclerosis-related genes in AD patients compared to placebo, these data suggest that modulation of vascular inflammation with systemic treatment should be explored in patients with AD.

Cross-sectional study of blood biomarkers of patients with moderate to severe alopecia areata reveals systemic immune and cardiovascular biomarker dysregulation.

BACKGROUND: Although there is increased understanding of the alopecia areata (AA) pathogenesis based on studies in scalp tissues, little is known about its systemic profile. OBJECTIVE: To evaluate the blood proteomic signature of AA and determine biomarkers associated with increased disease severity. METHODS: In a cross-sectional study, we assessed 350 inflammatory and cardiovascular proteins using OLINK high-throughput proteomics in patients with moderate to severe AA (n = 35), as compared with healthy individuals (n = 36), patients with moderate to severe psoriasis (n = 19), and those with atopic dermatitis (n = 49). RESULTS: Seventy-four proteins were significantly differentially expressed between AA and control individuals (false discovery rate, <.05) including innate immunity (interleukin [IL] 6/IL-8), T helper (Th) type 1 (interferon [IFN] γ/CXCL9/CXCL10/CXCL11), Th2 (CCL13/CCL17/CCL7), Th17 (CCL20/PI3/S100A12), and cardiovascular-risk proteins (OLR1/OSM/MPO/PRTN3). Eighty-six biomarkers correlated with AA clinical severity (P < .05), including Th1/Th2, and cardiovascular/atherosclerosis-related proteins, including SELP/PGLYRP1/MPO/IL-18/OSM (P < .05). Patients with AA totalis/universalis showed the highest systemic inflammatory tone, including cardiovascular risk biomarkers, compared to control individuals and even to patients with atopic dermatitis and those with psoriasis. The AA profile showed some Th1/Th2 differences in the setting of concomitant atopy. LIMITATIONS: Our analysis was limited to 350 proteins. CONCLUSION: This study defined the abnormalities of moderate to severe AA and associated circulatory biomarkers. It shows that AA has systemic immune, cardiovascular, and atherosclerosis biomarker dysregulation, suggesting the need for systemic treatment approaches.

Age-specific changes in the molecular phenotype of patients with moderate-to-severe atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) shows differential clinical presentation in older compared with younger patients. Nevertheless, changes in the AD molecular profile with age are unknown. OBJECTIVE: We sought to characterize age-related changes in the AD profile. METHODS: We evaluated age-specific changes in lesional and nonlesional tissues and blood from patients with moderate-to-severe AD (n = 246) and age-matched control subjects (n = 71) using immunohistochemistry, quantitative real-time PCR, and Singulex in a cross-sectional study. Patients were analyzed by age group (18-40, 41-60, and ≥61 years). RESULTS: Although disease severity/SCORAD scores were similar across AD age groups (mean, approximately 60 years; P = .873), dendritic cell infiltrates (CD1b+ and FcεRI+, P < .05) decreased with age. TH2 measures (IL5, IL13, CCL13, CCL18, and CCL26) significantly decreased with age in patients with AD, despite increasing with age in control subjects. Consistent with TH2 axis decreases, serum IgE levels and eosinophil counts negatively correlated with age in patients with AD (r = -0.24 and r = -0.23, respectively; P < .05). TH22-secreted IL22 expression levels also decreased with age uniquely in patients with AD (P < .05). Expression of TH1-related (IFNG, IL12/23p40, STAT1, and CXCL9; P < .05 for CXCL9) and TH17-related (IL17A and IL20; P < .05 for IL20) markers increased with age in both patients with AD and control subjects. Expression of terminal differentiation measures significantly increased in older patients with AD (loricrin [LOR] and filaggrin [FLG], P < .05), whereas expression of S100As (S100A8, P < .01) and hyperplasia markers (epidermal thickness, keratin 16, and Ki67; P < .05 for keratin 16) decreased. Serum trends in AD mimicked skin findings, with TH2 downregulation (CCL26; r = -0.32, P < .1) and TH1 upregulation (IFN-γ; r = 0.48, P < .01) with age. CONCLUSION: The adult AD profile varies with age. Although TH1/TH17 skewing increases in both patients with AD and control subjects, patients with AD show unique decreases in TH2/TH22 polarization and normalization of epithelial abnormalities. Thus age-specific treatment approaches might be beneficial for AD.

Atopic dermatitis in African American patients is TH2/TH22-skewed with TH1/TH17 attenuation.

BACKGROUND: African Americans (AA) are disproportionately impacted by atopic dermatitis (AD), with increased prevalence and therapeutic challenges unique to this population. Molecular profiling data informing development of targeted therapeutics for AD are derived primarily from European American (EA) patients. These studies are absent in AA, hindering development of effective treatments for this population. OBJECTIVE: We sought to characterize the global molecular profile of AD in the skin of AA patients as compared with that of EA AD and healthy controls. METHODS: We performed RNA-Seq with reverse transcription polymerase chain reaction validation and immunohistochemistry studies in lesional and nonlesional skin of AA and EA AD patients vs healthy controls. RESULTS: African American AD lesions were characterized by greater infiltration of dendritic cells (DCs) marked by the high-affinity immunoglobulin E (IgE) receptor (FcεR1+) compared with EA AD (P < .05). Both AD cohorts showed similarly robust up-regulation of Th2-related (CCL17/18/26) and Th22-related markers (interleukin [IL]-22, S100A8/9/12), but AA AD featured decreased expression of innate immune (tumor necrosis factor [TNF], IL-1ß), Th1-related (interferon gamma [IFN-γ], MX1, IL-12RB1), and Th17-related markers (IL-23p19, IL-36G, CXCL1) vs EA AD (P < .05). The Th2 (IL-13) and Th22-related products (IL-22, S100A8/9/12) and serum IgE were significantly correlated with clinical severity (Scoring of Atopic Dermatitis [SCORAD]) in AA. Fillagrin (FLG) was exclusively down-regulated in EA AD, whereas loricrin (LOR) was down-regulated in both AD cohorts and negatively correlated with SCORAD in AA. CONCLUSION: The molecular phenotype of AA AD skin is characterized by attenuated Th1 and Th17 but similar Th2/Th22-skewing to EA AD. Our data encourages a personalized medicine approach accounting for phenotype-specific characteristics in future development of targeted therapeutics and clinical trial design for AD.

N-Terminal Peptide Detection with Optimized Peptide-Spectrum Matching and Streamlined Sequence Libraries.

We identified tryptic peptides in yeast cell lysates that map to translation initiation sites downstream of the annotated start sites using the peptide-spectrum matching algorithms OMSSA and Mascot. To increase the accuracy of peptide-spectrum matching, both algorithms were run using several standardized parameter sets, and Mascot was run utilizing a, b, and y ions from collision-induced dissociation. A large fraction (22%) of the detected N-terminal peptides mapped to translation initiation downstream of the annotated initiation sites. Expression of several truncated proteins from downstream initiation in the same reading frame as the full-length protein (frame 1) was verified by western analysis. To facilitate analysis of the larger proteome of Drosophila, we created a streamlined sequence library from which all duplicated trypsin fragments had been removed. OMSSA assessment using this "stripped" library revealed 171 peptides that map to downstream translation initiation sites, 76% of which are in the same reading frame as the full-length annotated proteins, although some are in different reading frames creating new protein sequences not in the annotated proteome. Sequences surrounding implicated downstream AUG start codons are associated with nucleotide preferences with a pronounced three-base periodicity N1^G2^A3.

Atopic dermatitis stratification: current and future perspective on skin and blood transcriptomic and proteomic profiling.

INTRODUCTION: Atopic dermatitis (AD) is a common, chronic inflammatory skin disorder driven by an intricate interplay of genetic, environmental, and immunological factors. AREAS COVERED: As a clinically heterogenous condition, AD may be stratified into subtypes based on factors including, chronicity, immunoglobulin E levels, severity, age, and ethnicity. Transcriptomic and proteomic analyses in skin and blood help elucidate the underlying molecular mechanisms of these AD subtypes, referred to as AD endotypes. Further characterizing AD endotypes using reliable biomarkers can facilitate the development of more effective and personalized therapeutics and improve our tools for monitoring disease progression and therapeutic response across a diverse subset of patients. Here, we aim to provide perspective on the latest research regarding AD stratification using skin and blood-based studies and insight into the implications of these findings on the future of AD research and clinical practice. EXPERT OPINION: The precise stratification of AD endotypes will allow for the development of reliable biomarkers and a more personalized medical treatment approach. Clinical practice and trials will eventually be able to bridge clinical with molecular data to optimize individualized treatments and more effectively monitor treatment response.

A phase 2a randomized vehicle-controlled multi-center study of the safety and efficacy of delgocitinib in subjects with moderate-to-severe alopecia areata.

Alopecia areata/AA is an autoimmune cause of nonscarring hair loss. The pathogenesis of AA involves many immune axes, including Th1/Th2 pathways. Delgocitinib is a pan-Janus kinase/JAK inhibitor that broadly blocks pro-inflammatory cytokines and has been effective in other inflammatory skin conditions. Recent human studies/reports have shown that use of some systemic JAK inhibitors led to hair regrowth, suggesting this medication class as a potential therapy for AA. However, topical treatment is desirable due to potential systemic side effects. To assess the efficacy and safety of topical delgocitinib in AA, we conducted a double-blind, randomized, vehicle-controlled clinical trial in 31 moderate-to-severe AA patients that were randomized 2:1 to receive delgocitinib ointment 30 mg/g (n = 20) or ointment vehicle (n = 11) for 12 weeks. The primary endpoint was change in severity of Alopecia Tool/SALT score from baseline to week 12. The secondary endpoint included safety profile by reported adverse events. Twenty-three subjects completed the trial, with eight discontinuing mostly due to voluntary withdrawal. Ten patients receiving delgocitinib ointment and three patients receiving vehicle showed SALT score improvements after 12 weeks, but the mean percent SALT improvement at week 12 compared to baseline between the two arms was not significant (p = 0.92). Our study suggests that delgocitinib ointment is not effective in moderate-to-severe AA, likely due to its inability to penetrate sufficiently deeply into the dermis of the scalp, but larger studies are necessary to assess whether a different formulation of topical JAK inhibitors may be suitable to treat mild or more localized forms of AA.

COVID-19 Symptoms Are Attenuated in Moderate-to-Severe Atopic Dermatitis Patients Treated with Dupilumab.

BACKGROUND: In the SARS-CoV-2/COVID-19 pandemic, we need to understand the impact of immunomodulatory medications on COVID-19 symptom severity in patients with inflammatory diseases, including the type 2/Th2 polarized skin disease, atopic dermatitis (AD). OBJECTIVE: Because it is believed that type 1/Th1 immunity controls viral infections and that there is a Th1/Th2 counter-regulation, we hypothesized that Th2 targeting with the IL-4Rα-antagonist, dupilumab, in patients with moderate-to-severe AD would rebalance the Th1/Th2 axis, potentially leading to attenuated COVID-19 symptoms. METHODS: A total of 1237 patients with moderate-to-severe AD in the Icahn School of Medicine at Mount Sinai Department of Dermatology were enrolled in a registry. Patients were screened for COVID-19-related symptoms and assigned a severity score (asymptomatic [0]-fatal [5]). Scores were compared among 3 treatment groups: dupilumab (n = 632), other systemic treatments (n = 107), and limited/no treatment (n = 498). Demographic and comorbid covariates were adjusted by multivariate generalized logistic regression models. RESULTS: The dupilumab-treated group showed reduced incidence and severity of COVID-19 symptoms versus other treatment groups. Dupilumab-treated patients were less likely to experience moderate-to-severe symptoms versus patients on other systemics (P = .01) and on limited/no treatment (P = .04), and less likely to experience any symptoms versus patients on other systemics (P = .01). This effect was seen in our entire cohort and in the subgroup of patients with verified COVID-19 or high-risk exposure. CONCLUSIONS: Patients on dupilumab experienced less severe COVID-19 manifestations and lesser symptoms compared with patients on other systemics and on limited/no treatment. These results suggest that Th2 modulation with dupilumab may have a protective effect on anti-viral immune response in patients with AD.

Th2/Th1 Cytokine Imbalance Is Associated With Higher COVID-19 Risk Mortality.

A major component of COVID-19 severe respiratory syndrome is the patient's immune response to the SARS-CoV-2 virus and the consequential multi-organ inflammatory response. Several studies suggested a potential role of CD4+ T cells in COVID-19 severe respiratory syndrome. We first hypothesized that there is a type 2 helper (Th2)/type 1 helper (Th1) imbalance in older age, male, asthma, smokers, and high ACE2 expression phenotype in the airway of non-infected patients. Next, we hypothesized that a Th2/Th1 imbalance may predict higher mortality in COVID-19 infected hospitalized patients with and without patient reported current asthma. We first analyzed publicly available gene expression from the sputum of 118 moderate-to-severe asthma patients and 21 healthy controls, and from nasal epithelium of 26 healthy current smokers and 21 healthy never smokers. Secondly, we profiled 288 new serum proteomics samples measured at admission from patients hospitalized within the Mount Sinai Health System with positive SARS-CoV-2 infection. We first computed Th1 and Th2 pathway enrichment scores by gene set variation analysis and then compared the differences in Th2 and Th1 pathway scores between patients that died compared to those that survived, by linear regression. The level of Th2/Th1 imbalance, as determined by the enrichment score, was associated with age, sex, and ACE2 expression in sputum, and with active smoking status in nasal epithelium (p < 0.05). Th2/Th1 imbalance at hospital admission in sera of patients was not significantly associated with death from COVID-19 (p = 0.11), unless evaluated in the asthmatic strata (p = 0.01). Using a similar approach we also observed a higher Th17/Th1 cytokine imbalance in all deceased patients compared to those that survived (p < 0.001), as well as in the asthmatic strata only (p < 0.01). Th2/Th1 imbalance is higher in the sera of asthma patients at admission that do not survive COVID-19, suggesting that the Th2/Th1 interplay may affect patient outcomes in SARS-CoV2 infection. In addition, we report that Th17/Th1 imbalance is increased in all patients that die of COVID-19.

GCN sensitive protein translation in yeast.

Levels of protein translation by ribosomes are governed both by features of the translation machinery as well as sequence properties of the mRNAs themselves. We focus here on a striking three-nucleotide periodicity, characterized by overrepresentation of GCN codons and underrepresentation of G at the second position of codons, that is observed in Open Reading Frames (ORFs) of mRNAs. Our examination of mRNA sequences in Saccharomyces cerevisiae revealed that this periodicity is particularly pronounced in the initial codons-the ramp region-of ORFs of genes with high protein expression. It is also found in mRNA sequences immediately following non-standard AUG start sites, located upstream or downstream of the standard annotated start sites of genes. To explore the possible influences of the ramp GCN periodicity on translation efficiency, we tested edited ramps with accentuated or depressed periodicity in two test genes, SKN7 and HMT1. Greater conformance to (GCN)n was found to significantly depress translation, whereas disrupting conformance had neutral or positive effects on translation. Our recent Molecular Dynamics analysis of a subsystem of translocating ribosomes in yeast revealed an interaction surface that H-bonds to the +1 codon that is about to enter the ribosome decoding center A site. The surface, comprised of 16S/18S rRNA C1054 and A1196 (E. coli numbering) and R146 of ribosomal protein Rps3, preferentially interacts with GCN codons, and we hypothesize that modulation of this mRNA-ribosome interaction may underlie GCN-mediated regulation of protein translation. Integration of our expression studies with large-scale reporter studies of ramp sequence variants suggests a model in which the C1054-A1196-R146 (CAR) interaction surface can act as both an accelerator and braking system for ribosome translation.