Clinical severity classes in COVID-19 pneumonia have distinct immunological profiles, facilitating risk stratification by machine learning.

Objective: Clinical triage in coronavirus disease 2019 (COVID-19) places a heavy burden on senior clinicians during a pandemic situation. However, risk stratification based on serum biomarker bioprofiling could be implemented by a larger, nonspecialist workforce. Method: Measures of Complement Activation and inflammation in patientS with CoronAvirus DisEase 2019 (CASCADE) patients (n = 72), (clinicaltrials.gov: NCT04453527), classified as mild, moderate, or severe (by support needed to maintain SpO2 > 93%), and healthy controls (HC, n = 20), were bioprofiled using 76 immunological biomarkers and compared using ANOVA. Spearman correlation analysis on biomarker pairs was visualised via heatmaps. Linear Discriminant Analysis (LDA) models were generated to identify patients likely to deteriorate. An X-Gradient-boost (XGB) model trained on CASCADE data to triage patients as mild, moderate, and severe was retrospectively employed to classify COROnavirus Nomacopan Emergency Treatment for covid 19 infected patients with early signs of respiratory distress (CORONET) patients (n = 7) treated with nomacopan. Results: The LDA models distinctly discriminated between deteriorators, nondeteriorators, and HC, with IL-27, IP-10, MDC, ferritin, C5, and sC5b-9 among the key predictor variables during deterioration. C3a and C5 were elevated in all severity classes vs. HC (p < 0.05). sC5b-9 was elevated in the "moderate" and "severe" categories vs. HC (p < 0.001). Heatmap analysis shows a pairwise increase of negatively correlated pairs with IL-27. The XGB model indicated sC5b-9, IL-8, MCP1, and prothrombin F1 and F2 were key discriminators in nomacopan-treated patients (CORONET study). Conclusion: Distinct immunological fingerprints from serum biomarkers exist within different severity classes of COVID-19, and harnessing them using machine learning enabled the development of clinically useful triage and prognostic tools. Complement-mediated lung injury plays a key role in COVID-19 pneumonia, and preliminary results hint at the usefulness of a C5 inhibitor in COVID-19 recovery.

Atopic dermatitis and ocular allergy: common mechanisms and uncommon questions.

PURPOSE OF REVIEW: Atopic dermatitis (AD) and ocular allergy aka allergic eye disease (AED) are two common conditions that often coexist in patients. However, molecular connections between these two conditions are incompletely understood. While common etiologic components including Th2 immune signaling have been suggested for AD and AED, the mechanism how current Th2-targetd therapies (dupilumab, tralokinumab) for AD can augment conjunctivitis is not well understood. RECENT FINDINGS: Differentially regulated genes and pathways relevant for AD disease manifestation are known. In contrast, similar information is not yet available for AED, which could be largely addressed by emerging noninvasive ocular sampling techniques. Emerging evidence indicated a reduction in goblet cell number and mucin production in a subpopulation of AD patients with AD leading to adverse ocular outcomes, while other potential mechanisms could also be involved. Involvement of particular barrier function protein(s) in AED needs further investigation. SUMMARY: Modern cytokine-targeted therapies for AD showed elevated risk for developing conjunctivitis. Recently developed noninvasive sampling techniques should be leveraged to identify AD endotypes associated with AED and with dupilumab-associated ocular outcomes.

Treatment Outcome with High versus low-to-moderate Dosing of Corticosteroids in Early vis-a-vis Late-onset Hypoxic Cases of COVID-19: A Multicentric Retrospective Cohort Study.

Background: Corticosteroid dosing in COVID-19 cases associated with early-onset and late-onset hypoxia have not been separately explored. Methods: In this retrospective cohort study, we divided hypoxic COVID-19 cases into groups based on timing of initiation of corticosteroids relative to onset of symptoms; Group A (≤6th day), Group B (7th-9th day) and Group C (≥10th day), each group being sub-grouped into high and low-to-moderate dose corticosteroid recipients. Cox regression with propensity scoring was used to compare 28-day mortality between high and low-to-moderate dose recipients separately in Group A, Group B, Group C. Results: Among 505 patients included, propensity score matched Cox regression showed greater risk of all-cause mortality among high dose recipients in Group A [HR= 7.35, 95%CI 3.36-16.11, p-value<0·01, N=114] and Group B [HR=3.17, 95%CI 1.65-6.07, p-value<0·01, N=251]. In Group C, mortality was lowest [12.8% (18/140)] with no significant difference between sub-groups [HR=2.52, 95%CI 0.22-29.15, p-value=0.459, N=140]. Kruskal-Wallis Test between Group A, Group B and Group C for six pre-defined exposure variables showed significant differences for Neutrophil:Lymphocyte Ratio (NLR). Conclusion: When steroids were initiated early (owing to an earlier onset of hypoxic symptoms), a high dose of corticosteroid was associated with greater overall 28-day mortality compared to a low-to-moderate dose. NLR, a marker for individual immune response, varied between treatment groups.

The Utility of Resolving Asthma Molecular Signatures Using Tissue-Specific Transcriptome Data.

An integrative analysis focused on multi-tissue transcriptomics has not been done for asthma. Tissue-specific DEGs remain undetected in many multi-tissue analyses, which influences identification of disease-relevant pathways and potential drug candidates. Transcriptome data from 609 cases and 196 controls, generated using airway epithelium, bronchial, nasal, airway macrophages, distal lung fibroblasts, proximal lung fibroblasts, CD4+ lymphocytes, CD8+ lymphocytes from whole blood and induced sputum samples, were retrieved from Gene Expression Omnibus (GEO). Differentially regulated asthma-relevant genes identified from each sample type were used to identify (a) tissue-specific and tissue-shared asthma pathways, (b) their connection to GWAS-identified disease genes to identify candidate tissue for functional studies, (c) to select surrogate sample for invasive tissues, and finally (d) to identify potential drug candidates via connectivity map analysis. We found that inter-tissue similarity in gene expression was more pronounced at pathway/functional level than at gene level with highest similarity between bronchial epithelial cells and lung fibroblasts, and lowest between airway epithelium and whole blood samples. Although public-domain gene expression data are limited by inadequately annotated per-sample demographic and clinical information which limited the analysis, our tissue-resolved analysis clearly demonstrated relative importance of unique and shared asthma pathways, At the pathway level, IL-1b signaling and ERK signaling were significant in many tissue types, while Insulin-like growth factor and TGF-beta signaling were relevant in only airway epithelial tissue. IL-12 (in macrophages) and Immunoglobulin signaling (in lymphocytes) and chemokines (in nasal epithelium) were the highest expressed pathways. Overall, the IL-1 signaling genes (inflammatory) were relevant in the airway compartment, while pro-Th2 genes including IL-13 and STAT6 were more relevant in fibroblasts, lymphocytes, macrophages and bronchial biopsies. These genes were also associated with asthma in the GWAS catalog. Support Vector Machine showed that DEGs based on macrophages and epithelial cells have the highest and lowest discriminatory accuracy, respectively. Drug (entinostat, BMS-345541) and genetic perturbagens (KLF6, BCL10, INFB1 and BAMBI) negatively connected to disease at multi-tissue level could potentially repurposed for treating asthma. Collectively, our study indicates that the DEGs, perturbagens and disease are connected differentially depending on tissue/cell types. While most of the existing literature describes asthma transcriptome data from individual sample types, the present work demonstrates the utility of multi-tissue transcriptome data. Future studies should focus on collecting transcriptomic data from multiple tissues, age and race groups, genetic background, disease subtypes and on the availability of better-annotated data in the public domain.

Health Effects of Trimellitic Anhydride Occupational Exposure: Insights from Animal Models and Immunosurveillance Programs.

Acid anhydrides are used by chemical industries as plasticizers. Trimellitic acid (TMA) is an acid anhydride widely utilized in factories to produce paints, varnishes, and plastics. In addition to causing direct irritant effects, TMA can augment antibody responses in exposed factory workers leading to occupational asthma. Therefore, industries producing TMA have implemented occupational immunosurveillance programs (OISPs) to ensure early diagnosis and medical management, involving exposure reduction/ complete removal of sensitized workers from exposure areas. Multiple animal models (mice strains, rat stains, guinea pig, swine) with different exposure patterns (dermal, nasal, vapor inhalation exposures for different time frames) have been described to elucidate the pathophysiology of TMA exposure. In TMA factories, in spite of implementing advanced environmental controls and personal protective measures to limit exposure, workers become TMA-sensitized. Animal models revealed sIgG, sIgE, sIgA, and sIgM along with pulmonary lesions, cellular infiltrates, alveolar hemorrhage, and pneumonitis associated with TMA exposure. Molecular studies showed involvement of specific functional gene clusters related to cytokine and chemokine responses, lung remodeling, and arginase function. However, thus far, there is no evidence supporting fetotoxic or carcinogenic effects of TMA. OISP data showed IgG and IgE responses in exposed factory workers. Interestingly, timelines for detectable sIgG response, in conjunction with its magnitude, have been shown to be a predictor for future sIgE response. OISPs have been very successful so far at creating a healthy and safe working environment for TMA-exposed factory workers. Graphical Abstract Trimellitic Acid (TMA), used to produce paints, varnishes and plastics, can cause irritant-mediated and immune-mediated occupational health problems. NCBI pubmed search indicated that multiple animal models (different animal types, with chronic vs. acute exposure type, using TMA dust/suspension applied via dermal or other routes) have been used by investigators to elucidate the pathobiology of TMA-exposure. Several outcomes have been measured including humoral, lung/ airway, lymph nodes and dermal/ ear thickening responses. Studies on human subjects have been conducted mostly as parts of Occupational immunosurveillance programs (OISPs) implemented to identify TMA-sensitized workers (using ImmunoCAP and Skin prick testing), monitoring them longitudinally and their medical management including exposure reduction/ complete removal of sensitized workers from exposure areas. Clinical management also includes identification of irritant-induced and/ or immune-mediated outcomes of TMA occupational exposure. Collectively, these studies have led to important insights into the pathomechanism of TMA-exposure and have been very successful at creating a safe working environment for TMA-exposed factory workers.

Development of a progesterone-specific IgE assay for diagnosing patients with suspected progestogen hypersensitivity.

BACKGROUND: Progesterone hypersensitivity (PH) manifests as a spectrum of allergic symptoms during the luteal phase of the menstrual cycle. Confirming progesterone-specific immunoglobulin E (IgE; sIgE) by skin testing is unreliable because of irritant responses. OBJECTIVE: To develop a progesterone sIgE assay to assist in diagnosing PH. METHODS: A progesterone-bovine serum albumin (BSA) conjugate was characterized and used to analyze sera collected from women in our center with suspected PH in a 1-batch enzyme-linked immunosorbent assay (ELISA) to establish high, low and negative cut-points. Sera collected from healthy nonatopic female subjects and from women with classical PH symptoms were included as negative and positive controls, respectively. Values exceeding the average negative control (OD + 3× the standard deviation) were considered positive. These cut-points were subsequently used to establish positive and negative results for serum from women with suspected PH received from other centers. A subset of high positive sera was used for ELISA-inhibition and in a beta-hexosaminidase mediator release assay to evaluate the specificity and functional relevance of progesterone-specific serum IgE, respectively. The numbers of true negative, false negative, true positive, and false positive samples were determined. RESULTS: The direct progesterone sIgE ELISA results ranged from high positive to low positive and negative compared with healthy nonatopic control sera. Enzyme-linked immunosorbent assay inhibition and beta-hexosaminidase mediator release confirmed specificity and functional relevance of progesterone-sIgE, respectively. The sensitivity, specificity positive predictive values and negative predictive values were found to be 82%, 100%, 86%, and 100%, respectively, using the mediator release assay results as the gold standard. CONCLUSION: This assay has a good specificity and positive predictive value for screening women with suspected PH for progesterone sIgE.

Retinoic acid prevents immunogenicity of milk lipocalin Bos d 5 through binding to its immunodominant T-cell epitope.

The major cow's milk allergen Bos d 5 belongs to the lipocalin protein family, with an intramolecular pocket for hydrophobic ligands. We investigated whether Bos d 5 when loaded with the active vitamin A metabolite retinoic acid (RA), would elicit differential immune responses compared to the unloaded state. By in silico docking an affinity energy of -7.8 kcal/mol was calculated for RA into Bos d 5. Loading of RA to Bos d 5 could be achieved in vitro, as demonstrated by ANS displacement assay, but had no effect on serum IgE binding in tolerant or challenge-positive milk allergic children. Bioinformatic analysis revealed that RA binds to the immunodominant T-cell epitope region of Bos d 5. In accordance, Bos d 5 significantly suppressed the CD3+ CD4+ cell numbers, proliferative response and IL-10, IL-13 and IFN-γ secretion from stimulated human PBMCs only when complexed with RA. This phenomenon was neither associated with apoptosis of T-cells nor with the activation of Foxp3+ T-cells, but correlated likely with enhanced stability to lysosomal digestion due to a predicted overlap of Cathepsin S cleavage sites with the RA binding site. Taken together, proper loading of Bos d 5 with RA may suppress its immunogenicity and prevent its allergenicity.

Leveraging Multilayered "Omics" Data for Atopic Dermatitis: A Road Map to Precision Medicine.

Atopic dermatitis (AD) is a complex multifactorial inflammatory skin disease that affects ~280 million people worldwide. About 85% of AD cases begin in childhood, a significant portion of which can persist into adulthood. Moreover, a typical progression of children with AD to food allergy, asthma or allergic rhinitis has been reported ("allergic march" or "atopic march"). AD comprises highly heterogeneous sub-phenotypes/endotypes resulting from complex interplay between intrinsic and extrinsic factors, such as environmental stimuli, and genetic factors regulating cutaneous functions (impaired barrier function, epidermal lipid, and protease abnormalities), immune functions and the microbiome. Though the roles of high-throughput "omics" integrations in defining endotypes are recognized, current analyses are primarily based on individual omics data and using binary clinical outcomes. Although individual omics analysis, such as genome-wide association studies (GWAS), can effectively map variants correlated with AD, the majority of the heritability and the functional relevance of discovered variants are not explained or known by the identified variants. The limited success of singular approaches underscores the need for holistic and integrated approaches to investigate complex phenotypes using trans-omics data integration strategies. Integrating omics layers (e.g., genome, epigenome, transcriptome, proteome, metabolome, lipidome, exposome, microbiome), which often have complementary and synergistic effects, might provide the opportunity to capture the flow of information underlying AD disease manifestation. Overlapping genes/candidates derived from multiple omics types include FLG, SPINK5, S100A8, and SERPINB3 in AD pathogenesis. Overlapping pathways include macrophage, endothelial cell and fibroblast activation pathways, in addition to well-known Th1/Th2 and NFkB activation pathways. Interestingly, there was more multi-omics overlap at the pathway level than gene level. Further analysis of multi-omics overlap at the tissue level showed that among 30 tissue types from the GTEx database, skin and esophagus were significantly enriched, indicating the biological interconnection between AD and food allergy. The present work explores multi-omics integration and provides new biological insights to better define the biological basis of AD etiology and confirm previously reported AD genes/pathways. In this context, we also discuss opportunities and challenges introduced by "big omics data" and their integration.

Multiple Transcriptome Data Analysis Reveals Biologically Relevant Atopic Dermatitis Signature Genes and Pathways.

Several studies have identified genes that are differentially expressed in atopic dermatitis (AD) compared to normal skin. However, there is also considerable variation in the list of differentially expressed genes (DEGs) reported by different groups and the exact cause of AD is still not fully understood. Using a rank-based approach, we analyzed gene expression data from five different microarray studies, comprising a total of 127 samples and more than 250,000 transcripts. A total of 89 AD gene expression signatures '89ADGES', including FLG gene, were identified to show dysregulation consistently across these studies. Using a Support Vector Machine, we showed that the '89ADGES' discriminates AD from normal skin with 98% predictive accuracy. Functional annotation of these genes implicated their roles in immune responses (e.g., betadefensin, microseminoprotein), keratinocyte differentiation/epidermal development (e.g., FLG, CORIN, AQP, LOR, KRT16), inflammation (e.g., IL37, IL27RA, CCL18) and lipid metabolism (e.g., AKR1B10, FAD7, FAR2). Subsequently, we validated a subset of signature genes using quantitative PCR in a mouse model. Using a bioinformatic approach, we identified keratinocyte pathway over-represented (P = <0.0006) among the 89 signature genes. Keratinocytes are known to play a major role in barrier function due to their location in the epidermis. Our result suggests that besides immune- mediated pathway, skin barrier pathways such as the keratinocyte differentiation pathway play a key role in AD pathogenesis. A better understanding of the role of keratinocytes in AD will be important for developing novel "barrier therapy" for this disease.

Identification of Microcystis aeruginosa Peptides Responsible for Allergic Sensitization and Characterization of Functional Interactions between Cyanobacterial Toxins and Immunogenic Peptides.

BACKGROUND: The cyanobacterium species Microcystis aeruginosa produces microcystin and an array of diverse metabolites believed responsible for their toxicity and/or immunogenicity. Previously, chronic rhinitis patients were demonstrated to elicit a specific IgE response to nontoxic strains of M. aeruginosa by skin-prick testing, indicating that cyanobacteria allergenicity resides in a non-toxin-producing component of the organism. OBJECTIVES: We sought to identify and characterize M. aeruginosa peptide(s) responsible for allergic sensitization in susceptible individuals, and we investigated the functional interactions between cyanobacterial toxins and their coexpressed immunogenic peptides. METHODS: Sera from patients and extracts from M. aeruginosa toxic [MC(+)] and nontoxic [MC(-)] strains were used to test IgE-specific reactivity by direct and indirect ELISAs; 2D gel electrophoresis, followed by immunoblots and mass spectrometry (MS), was performed to identify the relevant sensitizing peptides. Cytotoxicity and mediator release assays were performed using the MC(+) and MC(-) lysates. RESULTS: We found specific IgE to be increased more in response to the MC(-) strain than the MC(+) strain. This response was inhibited by preincubation of MC(-) lysate with increasing concentrations of microcystin. MS revealed that phycocyanin and the core-membrane linker peptide are the responsible allergens, and MC(-) extracts containing these proteins induced ß-hexosaminidase release in rat basophil leukemia cells. CONCLUSIONS: Phycobiliprotein complexes in M. aeruginosa have been identified as the relevant sensitizing proteins. Our finding that allergenicity is inhibited in a dose-dependent manner by microcystin toxin suggests that further investigation is warranted to understand the interplay between immunogenicity and toxicity of cyanobacteria under diverse environmental conditions. CITATION: Geh EN, Ghosh D, McKell M, de la Cruz AA, Stelma G, Bernstein JA. 2015. Identification of Microcystis aeruginosa peptides responsible for allergic sensitization and characterization of functional interactions between cyanobacterial toxins and immunogenic peptides. Environ Health Perspect 123:1159-1166; http://dx.doi.org/10.1289/ehp.1409065.

Primary identification, biochemical characterization, and immunologic properties of the allergenic pollen cyclophilin cat R 1.

Cyclophilin (Cyp) allergens are considered pan-allergens due to frequently reported cross-reactivity. In addition to well studied fungal Cyps, a number of plant Cyps were identified as allergens (e.g. Bet v 7 from birch pollen, Cat r 1 from periwinkle pollen). However, there are conflicting data regarding their antigenic/allergenic cross-reactivity, with no plant Cyp allergen structures available for comparison. Because amino acid residues are fairly conserved between plant and fungal Cyps, it is particularly interesting to check whether they can cross-react. Cat r 1 was identified by immunoblotting using allergic patients' sera followed by N-terminal sequencing. Cat r 1 (∼ 91% sequence identity to Bet v 7) was cloned from a cDNA library and expressed in Escherichia coli. Recombinant Cat r 1 was utilized to confirm peptidyl-prolyl cis-trans-isomerase (PPIase) activity by a PPIase assay and the allergenic property by an IgE-specific immunoblotting and rat basophil leukemia cell (RBL-SX38) mediator release assay. Inhibition-ELISA showed cross-reactive binding of serum IgE from Cat r 1-allergic individuals to fungal allergenic Cyps Asp f 11 and Mala s 6. The molecular structure of Cat r 1 was determined by NMR spectroscopy. The antigenic surface was examined in relation to its plant, animal, and fungal homologues. The structure revealed a typical cyclophilin fold consisting of a compact ß-barrel made up of seven anti-parallel ß-strands along with two surrounding α-helices. This is the first structure of an allergenic plant Cyp revealing high conservation of the antigenic surface particularly near the PPIase active site, which supports the pronounced cross-reactivity among Cyps from various sources.