How to hit the allergy target: A critical appraisal of intralymphatic immunotherapy with practical recommendations on ultrasound-guided injections.

BACKGROUND: Intralymphatic immunotherapy (ILIT) represents a promising novel approach treating allergic diseases. However, no standardized procedures or recommendations have been established or reported, despite the recognized fact that treatment efficacy relies on the ability to inject the allergen intranodally. OBJECTIVE: We aim to provide a critical appraisal of ILIT as a method of allergen immunotherapy and to deliver practical recommendations for accurate ILIT. METHODS: One hundred and seventy-three ILIT injections were performed in 28 (47%) women and 32 (53%) men with median age of 29 years (21-59). The injections were ultrasound-guided and recorded for retrospective analysis with respect to injection location, needle visibility, medication release, and patient characteristics. RESULTS: The results show that the correct positioning of the needle within the lymph node (LN) was most critical. If the whole length of the needle bevel was not inserted into the LN, substance backflush into the interstitium was observed. Selecting a more superficial LN and inserting the needle at a smaller angle towards the LN significantly improved needle visibility in the ultrasound. Longitudinal results showed that continuous practice significantly correlated with improved needle visibility and more accurate ILIT injections. CONCLUSION: Based on our results and practical experience, we propose several recommendations for LN selection and the correct handling of ultrasound probe and needle. We are confident that ILIT standardization and training will be important as to meet the goals of good safety and efficacy of ILIT.

Airway and systemic biomarkers of health effects after short-term exposure to indoor ultrafine particles from cooking and candles - A randomized controlled double-blind crossover study among mild asthmatic subjects.

BACKGROUND: There is insufficient knowledge about the systemic health effects of exposure to fine (PM2.5) and ultrafine particles emitted from typical indoor sources, including cooking and candlelight burning. We examined whether short-term exposure to emissions from cooking and burning candles cause inflammatory changes in young individuals with mild asthma. Thirty-six non-smoking asthmatics participated in a randomized controlled double-blind crossover study attending three exposure sessions (mean PM2.5 µg/m3; polycyclic aromatic hydrocarbons ng/m3): (a) air mixed with emissions from cooking (96.1; 1.1), (b) air mixed with emissions from candles (89.8; 10), and (c) clean filtered air (5.8; 1.0). Emissions were generated in an adjacent chamber and let into a full-scale exposure chamber where participants were exposed for five hours. Several biomarkers were assessed in relation to airway and systemic inflammatory changes; the primary outcomes of interest were surfactant Protein-A (SP-A) and albumin in droplets in exhaled air - novel biomarkers for changes in the surfactant composition of small airways. Secondary outcomes included cytokines in nasal lavage, cytokines, C-reactive protein (CRP), epithelial progenitor cells (EPCs), genotoxicity, gene expression related to DNA-repair, oxidative stress, and inflammation, as well as metabolites in blood. Samples were collected before exposure start, right after exposure and the next morning. RESULTS: SP-A in droplets in exhaled air showed stable concentrations following candle exposure, while concentrations decreased following cooking and clean air exposure. Albumin in droplets in exhaled air increased following exposure to cooking and candles compared to clean air exposure, although not significant. Oxidatively damaged DNA and concentrations of some lipids and lipoproteins in the blood increased significantly following exposure to cooking. We found no or weak associations between cooking and candle exposure and systemic inflammation biomarkers including cytokines, CRP, and EPCs. CONCLUSIONS: Cooking and candle emissions induced effects on some of the examined health-related biomarkers, while no effect was observed in others; Oxidatively damaged DNA and concentrations of lipids and lipoproteins were increased in blood after exposure to cooking, while both cooking and candle emissions slightly affected the small airways including the primary outcomes SP-A and albumin. We found only weak associations between the exposures and systemic inflammatory biomarkers. Together, the results show the existence of mild inflammation following cooking and candle exposure.

EAACI Molecular Allergology User's Guide 2.0.

Since the discovery of immunoglobulin E (IgE) as a mediator of allergic diseases in 1967, our knowledge about the immunological mechanisms of IgE-mediated allergies has remarkably increased. In addition to understanding the immune response and clinical symptoms, allergy diagnosis and management depend strongly on the precise identification of the elicitors of the IgE-mediated allergic reaction. In the past four decades, innovations in bioscience and technology have facilitated the identification and production of well-defined, highly pure molecules for component-resolved diagnosis (CRD), allowing a personalized diagnosis and management of the allergic disease for individual patients. The first edition of the "EAACI Molecular Allergology User's Guide" (MAUG) in 2016 rapidly became a key reference for clinicians, scientists, and interested readers with a background in allergology, immunology, biology, and medicine. Nevertheless, the field of molecular allergology is moving fast, and after 6 years, a new EAACI Taskforce was established to provide an updated document. The Molecular Allergology User's Guide 2.0 summarizes state-of-the-art information on allergen molecules, their clinical relevance, and their application in diagnostic algorithms for clinical practice. It is designed for both, clinicians and scientists, guiding health care professionals through the overwhelming list of different allergen molecules available for testing. Further, it provides diagnostic algorithms on the clinical relevance of allergenic molecules and gives an overview of their biology, the basic mechanisms of test formats, and the application of tests to measure allergen exposure.

Glial fibrillary acidic protein is a robust biomarker in cerebrospinal fluid and peripheral blood after traumatic spinal cord injury: a prospective pilot study.

PURPOSE: Biochemical biomarkers to determine the injury severity and the potential for functional recovery of traumatic spinal cord injury (TSCI) are highly warranted; however, it remains to be clarified whether cerebrospinal fluid (CSF) or peripheral blood (PB) is the ideal sample media. This study aims to measure and compare biomarker concentrations in CSF and PB and to explore associations between biomarker concentrations and injury severity, i.e., American Spinal Injury Association (ASIA) Impairment Scale (AIS) grade, and biomarker concentrations and clinical outcome, i.e., AIS grade improvement and Spinal Cord Independent Measure version III (SCIM-III) score. METHODS: From 2018 to 2020, we conducted a single-center prospective pilot study of TSCI patients (n=15) and healthy controls (n=15). Sample collection and clinical outcome assessment were performed at median 13 h [IQR: 19], 9 days [IQR: 2], and 148 days [IQR: 49] after TSCI. Concentrations of neuron-specific enolase (NSE); glial fibrillary acid protein (GFAP); neurofilament light chain (NfL); interferon-γ (IFN-γ); interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, and IL-13; and tumor necrosis factor α (TNF-α) were measured and associated to clinical outcomes. RESULTS: The biomarker concentrations were higher in CSF than PB. CSF concentrations of GFAP, NSE, IFN-y, TNF-a, IL-2, IL-12p70, IL-4, IL-10, and IL-13 and PB concentrations of GFAP and IFN-y were significantly associated with AIS grade, but not with AIS grade improvement or SCIM-III score. CONCLUSIONS: Our results support GFAP as a potential diagnostic biomarker that may be measured in CSF as well as PB.

The inflammatory response and blood-spinal cord barrier integrity in traumatic spinal cord injury: a prospective pilot study.

PURPOSE: Triggering of inflammatory responses and disruption of blood-spinal cord barrier (BSCB) integrity are considered pivotal events in the pathophysiology of traumatic spinal cord injury (TSCI). Yet, these events are poorly understood and described in humans. This study aims to describe inflammatory responses and BSCB integrity in human TSCI. METHODS: Fifteen TSCI patients and fifteen non-TSCI patients were prospectively recruited from Aarhus University Hospital, Denmark. Peripheral blood (PB) and cerebrospinal fluid (CSF) were collected at median day 0 [IQR: 1], median day 9 [IQR: 2], and median day 148 [IQR: 49] after injury. PB and CSF were analyzed for immune cells by flow cytometry, cytokines by multiplex immunoassay, and BSCB integrity by IgG Index. RESULTS: Eleven TSCI patients completed follow-up. Results showed alterations in innate and adaptive immune cell counts over time. TSCI patients had significantly increased cytokine concentrations in CSF at the first and second follow-up, while only concentrations of interleukin (IL)-4, IL-8, and tumor necrosis factor-α remained significantly increased at the third follow-up. In PB, TSCI patients had significantly increased IL-6, IL-8, and IL-10 concentrations and significantly decreased interferon-γ concentrations at the first follow-up. Results further showed increased IgG Index indicative of BSCB disruption in seven TSCI patients at the first follow-up, five TSCI patients at the second follow-up, and two patients at the third follow-up. CONCLUSIONS: Our results suggest that TSCI mainly triggers innate inflammatory responses that resolves over time, although with some degree of non-resolving inflammation, particularly in CSF. Our results cannot confirm BSCB disruption in all TSCI patients.

Strengthening the case for intralymphatic immunotherapy.

PURPOSE OF REVIEW: Intralymphatic immunotherapy (ILIT) is a promising novel method of immunotherapy, that is short and convenient, and may be very effective. Results have been varied, and efforts to unravel the real value of the treatment are ongoing. Methods used to assess the effect in clinical trials have been so varied that it is difficult to compare studies with each other. RECENT FINDINGS: Some advances have been made; the importance of injecting into the lymph node has been illustrated, and treatment with a range of medicines has proven to be successful. In meta-analyses the treatment has been shown to have no serious side effects and to be an effective short term desensitizing agent. Now it remains to be shown that ILIT also has long-term effects of tolerance. Preliminary data suggest that there is a long-term effect. SUMMARY: Injecting allergen directly into a lymph node strengthens the protective immune response. ILIT is safe and induces desensitization and very likely also induces tolerance. Compliance will improve compared with other treatment forms. If ILIT holds its promise, it will become an attractive option for patients with allergy.

Cerebrospinal fluid and peripheral blood proteomics in Traumatic Spinal Cord Injury: A prospective pilot study.

•Proteomics enable profiling of inflammatory responses after spinal cord injury.•Proteins are differentially expressed over time.•Proteins are differentially expressed in cerebrospinal fluid and peripheral blood.•A poor relationship exists between protein expression and neurological outcome.

A novel functional mast cell assay for the detection of allergies.

BACKGROUND: Clinical management of allergic diseases has been hampered by the lack of safe and convenient tests to reliably identify culprit allergens and to closely follow changes in disease activity over time. Because allergy diagnosis is a complex and laborious multistep procedure, there is an urgent need for simpler but still functionally accurate ex vivo assays allowing objective diagnosis, substantiating treatment choices, and quantifying therapeutic responses. OBJECTIVE: In this study, we sought to develop a novel functional cell-based assay that relies on passive sensitization of allergic effector cells with patient serum, circumventing current limitations in allergy diagnosis. METHODS: We genetically engineered a conditional homeobox B8 (Hoxb8)-immortalized progenitor line from the bone marrow of mice that are transgenic for the human high-affinity IgE receptor (FcεRIα). These cells can be reproducibly differentiated into mature Hoxb8 mast cells within 5 days of culture in virtually unlimited numbers. RESULTS: We demonstrate that the established Hoxb8 mast cell assay can be used to accurately measure total IgE levels, identify culprit allergens, longitudinally monitor allergen-specific immunotherapy, and potentially determine the time point of tolerance induction upon allergen-specific immunotherapy in patients with allergy. To facilitate the analysis of large testing volumes, we demonstrate a proof-of-concept for a high-throughput screening application based on fluorescent cell barcoding using the engineered Hoxb8 mast cells. CONCLUSIONS: Our results indicate that this novel mast cell assay could represent a valuable tool to support clinicians in the identification of IgE-mediated allergies and in the quantification of treatment efficacy as well as duration of therapeutic response.

RORγt inhibitors block both IL-17 and IL-22 conferring a potential advantage over anti-IL-17 alone to treat severe asthma.

BACKGROUND: RORγt is a transcription factor that enables elaboration of Th17-associated cytokines (including IL-17 and IL-22) and is proposed as a pharmacological target for severe asthma. METHODS: IL-17 immunohistochemistry was performed in severe asthma bronchial biopsies (specificity confirmed with in situ hybridization). Primary human small airway epithelial cells in air liquid interface and primary bronchial smooth muscle cells were stimulated with recombinant human IL-17 and/or IL-22 and pro-inflammatory cytokines measured. Balb/c mice were challenged intratracheally with IL-17 and/or IL-22 and airway hyperreactivity, pro-inflammatory cytokines and airway neutrophilia measured. Balb/c mice were sensitized intraperitoneally and challenged intratracheally with house dust mite extract and the effect of either a RORγt inhibitor (BIX119) or an anti-IL-11 antibody assessed on airway hyperreactivity, pro-inflammatory cytokines and airway neutrophilia measured. RESULTS: We confirmed in severe asthma bronchial biopsies both the presence of IL-17-positive lymphocytes and that an IL-17 transcriptome profile in a severe asthma patient sub-population. Both IL-17 and IL-22 stimulated the release of pro-inflammatory cytokine and chemokine release from primary human lung cells and in mice. Furthermore, IL-22 in combination with IL-17, but neither alone, elicits airway hyperresponsiveness (AHR) in naïve mice. A RORγt inhibitor specifically blocked both IL-17 and IL-22, AHR and neutrophilia in a mouse house dust mite model unlike other registered or advanced pipeline modes of action. Full efficacy versus these parameters was associated with 90% inhibition of IL-17 and 50% inhibition of IL-22. In contrast, anti-IL-17 also blocked IL-17, but not IL-22, AHR or neutrophilia. Moreover, the deregulated genes in the lungs from these mice correlated well with deregulated genes from severe asthma biopsies suggesting that this model recapitulates significant severe asthma-relevant biology. Furthermore, these genes were reversed upon RORγt inhibition in the HDM model. Cell deconvolution suggested that the responsible cells were corticosteroid insensitive γδ-T-cells. CONCLUSION: These data strongly suggest that both IL-17 and IL-22 are required for Th2-low endotype associated biology and that a RORγt inhibitor may provide improved clinical benefit in a severe asthma sub-population of patients by blocking both IL-17 and IL-22 biology compared with blocking IL-17 alone.

Allergen immunotherapy: The growing role of observational and randomized trial "Real-World Evidence".

BACKGROUND: Although there is a considerable body of knowledge about allergen immunotherapy (AIT), there is a lack of data on the reliability of real-world evidence (RWE) in AIT, and consequently, a lack of information on how AIT effectively works in real life. METHODS: To address the current unmet need for an appraisal of the quality of RWE in AIT, the European Academy of Allergy and Clinical Immunology Methodology Committee recently initiated a systematic review of observational studies of AIT, which will use the RELEVANT tool and the Grading of Recommendations Assessment, Development and Evaluation approach (GRADE) to rate the quality of the evidence base as a whole. The next step will be to develop a broadly applicable, pragmatic "real-world" database using systematic data collection. Based on the current RWE base, and perspectives and recommendations of authorities and scientific societies, a hierarchy of RWE in AIT is proposed, which places pragmatic trials and registry data at the positions of highest level of evidence. KEY RESULTS: There is a need to establish more AIT registries that collect data in a cohesive way, using standardized protocols. CONCLUSIONS: This will provide an essential source of real-world data that can be easily shared, promoting evidence-based research and quality improvement in study design and clinical decision-making.

Basophil activation test: Mechanisms and considerations for use in clinical trials and clinical practice.

The basophil activation test (BAT) is a functional assay that measures the degree of degranulation following stimulation with allergen or controls by flow cytometry. It correlates directly with histamine release. From the dose-response curve resulting from BAT in allergic patients, basophil reactivity (%CD63+ basophils) and basophil sensitivity (EC50 or similar) are the main outcomes of the test. BAT takes into account all characteristics of IgE and allergen and thus can be more specific than sensitization tests in the diagnosis of allergic disease. BAT reduces the need for in vivo procedures, such as intradermal tests and allergen challenges, which can cause allergic reactions of unpredictable severity. As it closely reflects the patients' phenotype in most cases, it may be used to support the diagnosis of food, venom and drug allergies and chronic urticaria, to monitor the natural resolution of food allergies and to predict and monitor clinical the response to immunomodulatory treatments, such as allergen-specific immunotherapy and biologicals. Clinical application of BAT requires analytical validation, clinical validation, standardization of procedures and quality assurance to ensure reproducibility and reliability of results. Currently, efforts are ongoing to establish a platform that could be used by laboratories in Europe and in the USA for quality assurance and certification.

Intralymphatic immunotherapy improves grass pollen allergic rhinoconjunctivitis: A 3-year randomized placebo-controlled trial.

BACKGROUND: Allergic rhinoconjunctivitis is a global health problem. Different allergen immunotherapy regimes are marketed but have low adherence because they are expensive, complex, and time-consuming. New allergen immunotherapy forms are needed. OBJECTIVE: In a 3-year follow-up double-blind randomized placebo-controlled trial, we aimed to investigate the effect of intralymphatic allergen immunotherapy (ILIT). METHODS: Patients with grass pollen rhinoconjunctivitis were treated with 3 ILIT injections and an ILIT booster 1 year later, 3 ILIT injections and a placebo booster, or 3 placebo injections and a placebo booster. Primary outcome was improvement in a combined symptom and medication score (cSMS). A novel evaluation tool with a linear regression model of cSMS and grass pollen counts was developed. Secondary outcomes were changes in grass specific immunoglobulins and skin and nasal provocation tests to grass pollen. RESULTS: A total of 36 patients were included. Log10-transformed cSMS was reduced by 0.30 (95% CI, 0.11-0.49; P = .002), equaling 48.5% (95% CI, 24.5%-62%), in the entire 3-year follow-up period, significant only in the first follow-up season but not in the second and third seasons. The regression model showed a 37% (P < .001) reduction in cSMS. The booster injection 1 year later had no additional effect. Secondary, repeated measures of IgE and IgG4 to grass showed significant between-group difference and within-group change in the ILIT groups. No change in provocation test results was found. CONCLUSIONS: ILIT gives a substantial reduction in grass pollen allergy symptoms and use of rescue medication, significant in the first season after treatment. A booster injection had no additional effect.

Basophil sensitivity reflects long-term clinical outcome of subcutaneous immunotherapy in grass pollen-allergic patients.

BACKGROUND: Allergic rhinoconjunctivitis is a public health problem. Allergen Immunotherapy is an effective and safe treatment, that modifies the natural course of allergic disease and induces long-term tolerance. OBJECTIVE: To correlate basophil and antibody biomarkers of subcutaneous immunotherapy to clinical outcomes and cellular changes in target tissue. METHODS: Adults suffering from allergic rhinoconjunctivitis due to grass pollen allergy were randomized to receive subcutaneous immunotherapy (n = 18) or to an open control group (n = 6). Patients reported daily symptom and medication scores and weekly rhinitis related quality of life scores during four pollen seasons. Biomarkers were measured every 3 months for three years treatment and every 6 months in the follow-up year. Nasal and cutaneous allergen challenge tests were performed annually. Leukocyte subsets were assessed in nasal mucosa biopsies at baseline and after treatment. RESULTS: Subcutaneous immunotherapy led to a 447-fold decrease in basophil sensitivity during the first treatment year. This remained 100-fold lower than baseline during the 3 year-treatment period and 10-fold lower during the follow-up year (n = 18, P = .03). Decrease in basophil sensitivity after three weeks of treatment predicted long-term improvement in seasonal combined symptom and medication scores (á¿¥=-0.69, P = .0027) during three years of treatment. AUC of IgE-blocking factor correlated to nasal allergen challenge (á¿¥ = 0.63, P = .0012) and SPT (á¿¥ = 0.45, P = .03). Plasma cell numbers in the nasal mucosa increased during treatment (P = .02). CONCLUSION: Decrease in basophil sensitivity after three weeks of subcutaneous allergen immunotherapy predicted the clinical outcome of this treatment.

SARS-CoV-2 evades immune detection in alveolar macrophages.

Respiratory infections, like the current COVID-19 pandemic, target epithelial cells in the respiratory tract. Alveolar macrophages (AMs) are tissue-resident macrophages located within the lung. They play a key role in the early phases of an immune response to respiratory viruses. AMs are likely the first immune cells to encounter SARS-CoV-2 during an infection, and their reaction to the virus will have a profound impact on the outcome of the infection. Interferons (IFNs) are antiviral cytokines and among the first cytokines produced upon viral infection. In this study, AMs from non-infectious donors are challenged with SARS-CoV-2. We demonstrate that challenged AMs are incapable of sensing SARS-CoV-2 and of producing an IFN response in contrast to other respiratory viruses, like influenza A virus and Sendai virus, which trigger a robust IFN response. The absence of IFN production in AMs upon challenge with SARS-CoV-2 could explain the initial asymptotic phase observed during COVID-19 and argues against AMs being the sources of pro-inflammatory cytokines later during infection.

Generation of a Human Allergic Mast Cell Phenotype from CD133+ Stem Cells.

Cultured human mast cells are a useful tool for research into innate immune responses as well as allergic mechanisms. Mast cells cultured from peripheral blood can provide information on immune mechanisms of known, selected individuals. With the method presented here, eight million mast cells can be cultured from ca. one million stem cells purified from one unit (450 mL) of human peripheral blood. Culture with IgE and IL4 optimizes an allergic phenotype of the mast cells.

Comparison of Normal and Metaplastic Epithelium in Patients with Stable versus Persistently Symptomatic Severe Asthma Using Laser-Capture Microdissection and Data-Independent Acquisition-Mass Spectrometry.

A proportion of patients with severe asthma (SA) show poor responses to traditional asthma medications; however, it remains unknown why some patients remain persistently symptomatic. Our objective was to explore the use of laser-capture microdissection of specific epithelial structures combined with quantitative data-independent acquisition mass spectrometry to elucidate differences in protein composition in patients with SA with varying symptom control. Unbiased label-free quantitative proteome analyses were performed on laser-capture-microdissected areas of specific epithelial structures from patients with SA with varying degrees of symptom control. A total of 1993 stable SA and 1652 symptomatic SA proteins in normal epithelium and 1458 stable SA and 1647 symptomatic SA proteins in metaplastic epithelium were quantified. When comparing proteome profiles based on symptom control, 33 proteins in patients with stable SA (≥twofold change; P ≤ 0.05) and 13 proteins in patients with persistently symptomatic SA (≥twofold change; P ≤ 0.05) were enriched significantly. When comparing proteome profiles based on epithelial status, 21 proteins in normal epithelium (≥twofold change; P ≤ 0.05) and 6 proteins in metaplastic epithelium (≥twofold change; P ≤ 0.05) were enriched significantly. New treatment strategies are needed for patients with severe asthma and exploratory studies of unbiased nature such as this may help when searching for new mechanisms and potential targets involved in the disease pathology.

Endosomal signalling via exosome surface TGFß-1.

Extracellular vesicles such as exosomes convey biological messages between cells, either by surface-to-surface interaction or by shuttling of bioactive molecules to a recipient cell's cytoplasm. Here we show that exosomes released by mast cells harbour both active and latent transforming growth factor ß-1 (TGFß-1) on their surfaces. The latent form of TGFß-1 is associated with the exosomes via heparinase-II and pH-sensitive elements. These vesicles traffic to the endocytic compartment of recipient human mesenchymal stem cells (MSCs) within 60 min of exposure. Further, the exosomes-associated TGFß-1 is retained within the endosomal compartments at the time of signalling, which results in prolonged cellular signalling compared to free-TGFß-1. These exosomes induce a migratory phenotype in primary MSCs involving SMAD-dependent pathways. Our results show that mast cell-derived exosomes are decorated with latent TGFß-1 and are retained in recipient MSC endosomes, influencing recipient cell migratory phenotype. We conclude that exosomes can convey signalling within endosomes by delivering bioactive surface ligands to this intracellular compartment.

Human Mast Cell Sensitization with IgE Increases miRNA-210 Expression.

BACKGROUND: MicroRNAs (miRNAs) represent important post-transcriptional regulators with a dynamic expression profile during health and disease. OBJECTIVES: We explored the miRNA profile of human mast cells (MCs) during sen-sitization with IgE, during activation through IgE, and relat ed it to prostaglandin D2 synthesis and histamine release. METHOD: We investigated the expression pattern of 762 miRNAs during the IgE-mediated sensitization and activation of MCs cultured from CD133+ stem cells that were isolated from allergic asthmatic patients and nonatopic controls. RESULTS: IgE-mediated sensitization increased the expression of miRNA-210 eight-fold. This increase was sustained during IgE-mediated MC activation. Furthermore, we confirmed the increase of the miRNA-132/212 cluster after MC activation. Predicted target genes of miRNA-210/132/212 were enriched in several pathways known to be involved in MC activation. Histamine release was significantly higher in MCs from allergic patients when compared to controls, and a number of miRNAs correlated with histamine release and prostaglandin D2 synthesis during MC activation. CONCLUSION: The miRNAs and analysis presented here can help to elucidate the role of miRNAs in mediator release during MC activation. We speculate that miRNA-210 could be important in MC sensitization that leads to allergic symptoms.