Topical corticosteroids inhibit allergic skin inflammation but are ineffective in impeding the formation and expansion of resident memory T cells.

BACKGROUND: Tissue-resident memory T (TRM ) cells are detrimental in allergic contact dermatitis (ACD), in which they contribute to the chronicity and severity of the disease. METHODS: We assessed the impact of a standard topical corticosteroid (TCS) treatment, triamcinolone acetonide (TA), on the formation, maintenance and reactivation of epidermal TRM cells in a preclinical model of ACD to 2,4-dinitrofluorobenzene. TA 0.01% was applied at different time points of ACD response and we monitored skin inflammation and tracked CD8+ CD69+ CD103+ TRM by flow cytometry and RNA sequencing. RESULTS: The impact of TA on TRM formation depended on treatment regimen: (i) in a preventive mode, that is, in sensitized mice before challenge, TA transiently inhibited the infiltration of effector T cells and the accumulation of TRM upon hapten challenge. In contrast, (ii) in a curative mode, that is, at the peak of the ACD response, TA blocked skin inflammation but failed to prevent the formation of TRM . Finally, (iii) in a proactive mode, that is, on previous eczema lesions, TA had no effect on the survival of skin TRM , but transiently inhibited their reactivation program upon allergen reexposure. Indeed, specific TRM progressively regained proliferative functions upon TA discontinuation and expanded in the tissue, leading to exaggerated iterative responses. Interestingly, TRM re-expansion correlated with the decreased clearance of hapten moieties from the skin induced by repeated TA applications. CONCLUSIONS: Our results demonstrate that TCS successfully treat ACD inflammation, but are mostly ineffective in impeding the formation and expansion of allergen-specific TRM , which certainly restricts the induction of lasting tolerance in patients with chronic dermatitis.

A breakthrough of immunoassay format for hapten: recent insights into noncompetitive immunoassays to detect small molecules.

Immunoassay based on the antibodies specific for targets has advantages of high sensitivity, simplicity and low cost, therefore it has received more attention in recent years, especially for the rapid detection of small molecule chemicals present in foods, diagnostics and environments. However, limited by low molecular weight and only one antigenic determinant existed, immunoassays for these small molecule chemicals, namely hapten substances, were commonly performed in a competitive immunoassay format, whose sensitivities were obviously lower than the sandwich enzyme-linked immunosorbent assay generally adaptable for the protein targets. In order to break through the bottleneck of detection format, researchers have designed and established several novel noncompetitive immunoassays for the haptens in the past few years. In this review, we focused on the four representative types of noncompetitive immunoassay formats and described their characteristics and applications in rapid detection of small molecules. Meanwhile, a systematic discussion on the current technologies challenges and the possible solutions were also summarized. This review aims to provide an updated overview of the current state-of-the-art in noncompetitive immunoassay for small molecules, and inspire the development of novel designs for small molecule detection.

Reconstructed human epidermis-based testing strategy of skin sensitization potential and potency classification using epidermal sensitization assay and in silico data.

The hazards and potency of skin sensitizers are traditionally determined using animal tests such as the local lymph node assay (LLNA); however, significant progress has been made in the development of non-animal test methods addressing the first three mechanistic key events of adverse outcome pathway in skin sensitization. We developed the epidermal sensitization assay (EpiSensA), which is a reconstructed human epidermis-based assay, by measuring four genes related to critical keratinocyte responses during skin sensitization. Four in vitro skin sensitization test methods (EpiSensA, direct peptide reactivity assay [DPRA], KeratinoSens™, and human cell line activation test [h-CLAT]) were systematically evaluated using 136 chemicals including lipophilic chemicals and pre/pro-haptens, which may be related to assay-specific limitations. The constructed database included existing and newly generated data. The EpiSensA showed a broader applicability domain and predicted the hazards with 82.4% and 78.8% accuracy than LLNA and human data. The EpiSensA could detect 76 out of 88 sensitizers at lower concentrations than the LLNA, indicating that the EpiSensA has higher sensitivity for the detection of minor sensitizing constituents. These results confirmed the potential use of the EpiSensA in evaluating a mixture of unknown compositions that can be evaluated by animal tests. To combine different information sources, the reconstructed human epidermis-based testing strategy (RTS) was developed based on weighted multiple information from the EpiSensA and TImes MEtabolism Simulator platform for predicting Skin Sensitization (TIMES-SS; RTSv1) or Organization for Economic Cooperation and Development (OECD) QSAR Toolbox automated workflow (RTSv2). The predictivities of the hazards and Globally Harmonized System (GHS) subcategories were equal to or better than the defined approaches (2 out of 3, integrated testing strategy [ITS]v1, and ITSv2) adopted as OECD Guideline 497.

Tolerogenic phenotype of dendritic cells is induced after hapten sensitization followed by attenuated contact hypersensitivity response in atopic dermatitis model NC/Nga mice.

Allergic contact dermatitis (ACD) and atopic dermatitis (AD) are common inflammatory diseases. We previously reported attenuated contact hypersensitivity (CHS) responses in AD model mice using 2,4-dinitrofluorobenzene, reflecting clinical experiments. However, previous studies have not addressed the commonality of findings across haptens and mechanisms focused on dendritic cells (DCs). Thus, this study evaluated CHS responses to fluorescein isothiocyanate (FITC) and DC migration and maturation in the sensitization phase of CHS in AD. CHS responses to FITC were compared between NC/Nga mice without and with AD induction (non-AD and AD mice, respectively). T-cell responses and DC migration and maturation after FITC-induced sensitization were examined in the draining lymph nodes of non-AD and AD mice. AD mice demonstrated reduced CHS responses to FITC under decreased T-cell proliferation following sensitization and interferon-γ production by hapten-specific T cells compared with non-AD mice. In addition, the number of FITC+CD11c+MHC class IIhigh migratory DCs 24 h after FITC sensitization was comparable between non-AD and AD mice. However, FITC+CD11c+MHC class IIhigh migratory DCs in AD mice exhibited lower expression levels of CD80 and CD86 and higher expression levels of PD-L1 and mRNA of transforming growth factor beta than non-AD mice. These findings suggest that attenuated CHS responses may be hapten-independent and the induction of the tolerogenic phenotype of hapten-bearing DCs can contribute to reduced T-cell proliferation after sensitization and CHS responses in AD.

The specific biopanning of single-domain antibody against haptens based on a functionalized cryogel.

Phage display technology is commonly applied for high-throughput screening of single-domain antibodies (sdAbs), and the problem of non-specific adsorption caused by carrier proteins seriously affects the biopanning of single-domain antibodies specific to haptens. In this paper, enrofloxacin (ENR)-functionalized cryogels were prepared by the ethylenediamine (EDA) and carbodiimide methods for application in the biopanning of ENR-specific phages. To improve the efficiency of biopanning, double blocking, a wash solution flow rate of 1 mL/min, and phage pre-incubation were applied to the biopanning process through single-factor experiments. Results of flat colony counting showed that the phage output of AG-ENR cryogels was 15 times higher than that of AG cryogels for the same input amount. And seven complete sequences of ENR-specific shark sdAbs were obtained by monoclonal phage ELISA and sequence alignment. All these results indicate that functionalized cryogels could be used as a novel and efficient method for phage biopanning for single-domain antibodies to haptens.

Advances in atopic dermatitis in 2019-2020: Endotypes from skin barrier, ethnicity, properties of antigen, cytokine profiles, microbiome, and engagement of immune cells.

Key research advances in atopic dermatitis (AD) suggest the complexity of its endotypes. A comprehensive serum biomarker panel revealed at least 4 types of AD. Some represent classic TH2-dominant AD with filaggrin mutations commonly reported in Europeans, a simultaneously activated multipolar axis of cytokines often reported in Asian individuals, and an intrinsic type characterized by TH2 inferiority. Innate lymphoid cells, including natural killer cells, natural killer T cells, and fibroblasts, play a role in AD development and heterogeneity. Here, we discuss the endotypes of AD from the perspective of antigen types (hapten vs protein antigens), barrier function, and a novel set of immune cells. Endotypic stratification of AD may lead to the development of customized therapeutic strategies in the future.

[Extended understanding of pathogenesis and treatment of contact allergy]. / Erweitertes Verständnis von Pathogenese und Therapie der Kontaktallergie.

BACKGROUND: While the pathogenesis of contact allergy in recent years has increasingly focused on the mechanisms of the innate immune response, valid therapeutic options are still lacking. AIMS: This article intends to shed light on the background of contact allergy development as well as possible risk factors and to highlight potential new therapeutic options. MATERIALS AND METHODS: Allergic contact dermatitis (ACD) as well as the sensitization and trigger phase, potential risk factors as well as the therapy options including (current) PubMed-listed literature are described. RESULTS: Inflammation plays a central role in ACD. The innate immune system responds to contact allergens as well as to infection. Elucidation of the mechanisms will enable a targeted therapeutic intervention in the future. CONCLUSION: Although there is still a need for research, many parts of the contact allergy pathogenesis are now better understood. In particular, the essential role of the innate immune response not only for the sensitization but also for the elicitation phase seems to be established. Implementation of today's knowledge into new therapeutic approaches and their application testing remains important and exciting.

Overexpression of O-polysaccharide chain length regulators in Gram-negative bacteria using the Wzx-/Wzy-dependent pathway enhances production of defined modal length O-polysaccharide polymers for use as haptens in glycoconjugate vaccines.

AIMS: O-polysaccharide (OPS) molecules are protective antigens for several bacterial pathogens, and have broad utility as components of glycoconjugate vaccines. Variability in the OPS chain length is one obstacle towards further development of these vaccines. Introduction of sizing steps during purification of OPS molecules of suboptimal or of mixed lengths introduces additional costs and complexity while decreasing the final yield. The overall goal of this study was to demonstrate the utility of engineering Gram-negative bacteria to produce homogenous O-polysaccharide populations that can be used as the basis of carbohydrate vaccines by overexpressing O-polysaccharide chain length regulators of the Wzx-/Wzy-dependent pathway. METHOD AND RESULTS: The O-polysaccharide chain length regulators wzzB and fepE from Salmonella Typhimurium I77 and wzz2 from Pseudomonas aeruginosa PAO1 were cloned and expressed in the homologous organism or in other Gram-negative bacteria. Overexpression of these Wzz proteins in the homologous organism significantly increased the proportion of long or very long chain O-polysaccharides. The same observation was made when wzzB was overexpressed in Salmonella Paratyphi A and Shigella flexneri, and wzz2 was overexpressed in two other strains of P. aeruginosa. CONCLUSIONS: Overexpression of Wzz proteins in Gram-negative bacteria using the Wzx/Wzy-dependant pathway for lipopolysaccharide synthesis provides a genetic method to increase the production of an O-polysaccharide population of a defined size. SIGNIFICANCE AND IMPACT OF THE STUDY: The methods presented herein represent a cost-effective and improved strategy for isolating preferred OPS vaccine haptens, and could facilitate the further use of O-polysaccharides in glycoconjugate vaccine development.

Expanding the Genetic Code for a Dinitrophenyl Hapten.

Haptens, such as dinitrophenyl (DNP) are small molecules that induce strong immune responses when attached to proteins or peptides and, as such, have been exploited for diverse applications. We engineered a Methanosarcina barkeri pyrrolysyl-tRNA synthetase (mbPylRS) to genetically encode a DNP-containing unnatural amino acid, N(6) -(2-(2,4-dinitrophenyl)acetyl)lysine (DnpK). Although this moiety was unstable in Escherichia coli, we found that its stability was enhanced in mammalian HEK 293T cells and was able to induce selective interactions with anti-DNP antibodies. The capability of genetically introducing DNP into proteins is expected to find broad applications in biosensing, immunology, and therapeutics.

Elevated levels of antibodies against xenobiotics in a subgroup of healthy subjects.

In spite of numerous research efforts, the exact etiology of autoimmune diseases remains largely unknown. Genetics and environmental factors, including xenobiotics, are believed to be involved in the induction of autoimmune disease. Some environmental chemicals, acting as haptens, can bind to a high-molecular-weight carrier protein such as human serum albumin (HSA), causing the immune system to misidentify self-tissue as an invader and launch an immune response against it, leading to autoimmunity. This study aimed to examine the percentage of blood samples from healthy donors in which chemical agents mounted immune challenges and produced antibodies against HSA-bound chemicals. The levels of specific antibodies against 12 different chemicals bound to HSA were measured by ELISA in serum from 400 blood donors. We found that 10% (IgG) and 17% (IgM) of tested individuals showed significant antibody elevation against aflatoxin-HSA adduct. The percentage of elevation against the other 11 chemicals ranged from 8% to 22% (IgG) and 13% to 18% (IgM). Performance of serial dilution and inhibition of the chemical-antibody reaction by specific antigens but not by non-specific antigens were indicative of the specificity of these antibodies. Although we lack information about chemical exposure in the tested individuals, detection of antibodies against various protein adducts may indicate chronic exposure to these chemical haptens in about 20% of the tested individuals. Currently the pathological significance of these antibodies in human blood is still unclear, and this protein adduct formation could be one of the mechanisms by which environmental chemicals induce autoimmune reactivity in a significant percentage of the population.

Synthesis and Use of a Phosphonate Amidine to Generate an Anti-Phosphoarginine-Specific Antibody.

Protein arginine phosphorylation is a post-translational modification (PTM) that is important for bacterial growth and virulence. Despite its biological relevance, the intrinsic acid lability of phosphoarginine (pArg) has impaired studies of this novel PTM. Herein, we report for the first time the development of phosphonate amidines and sulfonate amidines as isosteres of pArg and then use these mimics as haptens to develop the first high-affinity sequence independent anti-pArg specific antibody. Employing this anti-pArg antibody, we further showed that arginine phosphorylation is induced in Bacillus subtilis during oxidative stress. Overall, we expect this antibody to see widespread use in analyzing the biological significance of arginine phosphorylation. Additionally, the chemistry reported here will facilitate the generation of pArg mimetics as highly potent inhibitors of the enzymes that catalyze arginine phosphorylation/dephosphorylation.

Synthesis of haptens and development of an indirect enzyme-linked immunosorbent assay for tris(2,3-dibromopropyl) isocyanurate.

A competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed for detection of tris(2,3-dibromopropyl) isocyanurate (TBC). Polyclonal antibodies against TBC were raised from synthesized haptens and then screened against various coating antigens. After optimization of the immunoassay conditions, the linear range and IC50 value of the assay were 0.30-100 and 5.17 µg/L, respectively, with little cross-reactivity (≤2%). Recovery of various samples (water, serum, soil) was tested and the values ranged from 68% to 110%. This ciELISA was also applied to determine TBC in the riverside soil of the Liuyang River, and the results were compared with the data obtained by UHPLC-MS/MS. The experimental assay results confirmed that this proposed immunoassay is a specific, sensitive, and reliable method for determination and monitoring of TBC.

Contact urticaria syndrome caused by haptens.

In the group of urticaria, contact urticaria syndrome is a particular variety. In these patients, appearance of typical skin lesions is preceded by contact of the skin and mucous membranes with various inhaled allergens, nutrients or contact details. Furthermore, symptoms connected with contact urticaria syndrome are characterized by gradual, stepwise waveform, which can be easily generalized - patients may develop systemic symptoms similar to those found in the angioedema, asthma or anaphylactic shock. It is an attribute of contact urticaria syndrome in the course of which potentially life-threatening symptoms may develop after contact of the skin with the allergen. The underlying mechanisms are poorly understood; both immunological and non-immunological mechanisms are taken into account, therefore contact urticaria syndrome can be classified into two categories - allergic and non-allergic. An intriguing phenomenon seems to be the immediate reaction after exposure to low molecular weight allergens - haptens, such as metals, which are usually the cause of delayed allergic reactions. Diagnosis is based on clinical presentation indicating a coincidence of the onset of allergy with contract with allergen, and helpful exposure tests. Treatment consists of supportive antihistamines and corticosteroids - locally and systemically. In the case of anaphylaxis, appropriate treatment intensification of the integration of pressor amines and hydration is necessary. It is also regarded that prevention is advisable, which consists of relevant information to avoid situations connected with contact with well-known factors. In this paper we describe a case of a 57-year-old female admitted to the Department of Internal Medicine, Geriatrics and Allergology, Medical University in Wroclaw to undergo diagnostic tests of chronic urticaria and angioedema. According to information obtained from the clinical presentation and after the diagnostic procedures, contact urticaria syndrome due to exposure to metals was diagnosed.

[Lugol as a rare cause of anaphylactic reaction after colposcopy. Case report]. / Lugol como causa inusual de anafilaxia después de la colposcopia. Reporte de un caso clínico.

INTRODUCTION: Lugol is a solution composed of elemental iodine (5%) and potassium iodide (10%) together with distilled water, used during colposcopic assessment to identify possible cervical cell alterations. CASE REPORT: A 31-year-old female who presents an episode suggestive of anaphylaxis ninety minutes after a colposcopy exploration, successfully treated with intramuscular hydrocortisone and desclorfeniramine. During colposcopy Lugol solution and acetic acid was applied. Skin prick test (SPT) with Lugol solution was positive (papule 9x7 mm). Four control tests were negative. Intradermal tests (IDT) were positive with Lugol and elemental iodine, the last one turned-out irritant. It was ruled out possible cross-reactivity with other iodine preparations (Betadine®) and potassium iodide (Yoduk®). CONCLUSIONS: Our report demonstrates a rare case of allergy to Lugol solution with positive SPT and a clinical suggestive reaction, with tolerance to other iodine preparations and potassium iodide.

INTRODUCCIÓN: El lugol es una solución compuesta por yodo elemental (5%), yoduro de potasio (10%) y agua destilada, utilizada durante las colposcopias para detectar alteraciones celulares en el cérvix. REPORTE DE CASO: Paciente femenina de 31 años, que tuvo un evento de anafilaxia noventa minutos después de la colposcopia, tratada exitosamente con hidrocortisona por vía intramuscular y desclorfeniramina. Durante la colposcopia se aplicó lugol y ácido acético. La prueba de prick con lugol resultó positiva, con formación de una pápula de 9 x 7 mm, al igual que las pruebas intradérmicas para lugol y yodo elemental. Cuatro controles fueron negativos, excepto para yodo elemental, que resultó irritante en intradermorreacción. Se descartó reactividad cruzada con otras soluciones yodadas (Betadine®) y (Yoduk®). CONCLUSIONES: Reportamos un caso raro de alergia a lugol con prick positivo y una reacción clínica sugerente, con tolerancia a otras preparaciones yodadas y a yoduro de potasio.

Unique protein signatures evolve during the course of a delayed-type hypersensitivity reaction in human skin.

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.

Portrait of an autologous cancer vaccine: Then and now.

Active immunotherapy of cancer with therapeutic vaccines has been the subject of experimental and clinical studies for at least 50 years. Our approach has employed 1) autologous, human cancer cells because of extensive evidence that tumor rejection antigens may differ between multiple tumors of the same histology; 2) the immunopotentiating drug, cyclophosphamide; and 3) haptens, particularly dinitrophenyl. Multiple clinical trials in 455 patients with melanoma and ovarian cancer have shown that administration of haptenized vaccines at the proper dosage-schedule regularly induces T cell-mediated immunity to autologous tumor cells as measured by delayed-type hypersensitivity. Moreover, the vaccine causes changes in the tumor site suggestive of an immune reaction, including inflammation and infiltration with CD8+ T lymphocytes that are activated and produce cytokines. The T cell response is oligoclonal, and dominant Vß families differ between patients. Studies of measurable metastases show clinically important tumor regression. Commercial development of this technology is clearly feasible.

Detection of ustilaginoidins in rice samples by immunoassay based on monoclonal antibodies prepared from hemiustilaginoidin-derived haptens.

Ustilaginoidins are a class of bis-naphtho-γ-pyrone mycotoxins to threaten humans, animals and environment. Ustilaginoidins are produced by Villosiclava virens, the rice false smut pathogen. To prepare antibodies for quantitatively analyzing ustilaginoidins in rice samples, hemiustilaginoidins D and F from the laccase gene deficiency mutant of V. virens respectively reacted with diazonium 4-aminobenzoic acid to obtain haptens with a carboxyl group, which further reacted with bovine serum albumin or ovalbumin to get their complete antigens. Two monoclonal antibodies (mAbs) designated as 4A12C6 and 5F4F6 were developed by immunization. The relationships between mAb sensitivity and 20 ustilaginoidins were described. 4A12C6 was chosen for further analysis as it could recognize main ustilaginoidins and was more sensitive than 5F4F6. The achieved indirect competitive enzyme-linked immunosorbent assay (icELISA) based on 4A12C6 had a half maximal inhibitory concentration (IC50) of 0.76 ng/mL and working range of 0.2-2.8 ng/mL to ustilaginoidin A. The results of ustilaginoidins-contaminated rice samples by icELISA detection were consistent with those determined by HPLC‒DAD detection. Therefore, we developed a new strategy to get haptens from the biosynthetic precursors with half structures of ustilaginoidins. The achieved icELISA was demonstrated as a convenient method to monitor ustilaginoidin content in rice samples, and showed that the contents of total ustilaginoidins from the rice cultivars with low resistance to rice false smut were more than those of high resistance cultivars.

Multiplex Immunochromogenic Tissue Staining Employing Primary Antibodies from the Same Species.

Chromogenic immunohistochemistry (IHC) serves as an essential assay for the diagnoses of many diseases including cancer. Single-marker IHC detection is the standard used for clinical diagnostic assays. A technology to stain multiple biomarkers chromogenically on a single tissue will also yield contextual biomarker information. Methods to chromogenically stain multiple biomarkers simultaneously employing antibodies from the same species are limited and require complex protocols. Here we describe both manual and automated protocols using the UltraPlex™ mxIHC technology that allows simultaneous detection of up to three biomarkers on a single tissue using a single heat-induced antigen retrieval step in formaldehyde-fixed paraffin-embedded (FFPE) tissue and using primary antibodies from any species.

Lateral flow immunoassays for antigens, antibodies and haptens detection.

Lateral flow immunoassay (LFIA) is widely used as a rapid point-of-care testing (POCT) technique in food safety, veterinary and clinical detection on account of the accessible, fast and low-cost characteristics. After the outbreak of the coronavirus disease 2019 (COVID-19), different types of LFIAs have attracted considerable interest because of their ability of providing immediate diagnosis directly to users, thereby effectively controlling the outbreak. Based on the introduction of the principles and key components of LFIAs, this review focuses on the major detection formats of LFIAs for antigens, antibodies and haptens. With the rapid innovation of detection technologies, new trends of novel labels, multiplex and digital assays are increasingly integrated with LFIAs. Therefore, this review will also introduce the development of new trends of LFIAs as well as its future perspectives.