In the newly published article: “Medical algorithm: Diagnosis and treatment of radiocontrast media hypersensitivity” K. Brockow [1], suggested an algorithm for the evaluation of hypersensitivity to radiocontrast media (RCM). However, one important aspect has been left out of the suggested algorithm, the opportunity to desensitize patients with probable or confirmed immediate hypersensitivity reactions to radiocontrast media.  Especially those reacting to more than one substance. Desensitization to allergens is a standard preventative measure of anaphylaxis when patients have a history of severe immediate hypersensitivity reactions to a drug, and there are no therapeutic alternatives [2]. Protocols for RCM desensitization are known from case reports [3,4], and need more work and research before standardized protocols can be recommended, but they can facilitate the use of radiocontrast media in important situations such as the need for acute coronary angiograms. Such situations are rare, but an algorithm should include information and pathways to facilitate the best treatment possible for all patients. Suggested change in the algorithm is to include the option “rapid desensitization” in the pathway “RCM urgently needed without test possibility and indispensable need for the RCM”, and in the pathway for ”patients with confirmed RCM hypersensitivity with immediate hypersensitivity reactions to several RCM’s for situations with indispensable need for RCM”. This would help to facilitate the best point of care for more patients with hypersensitivity to RCM. Dr. Alnæs has nothing to disclose. Dr Alnæs is the only contributor to this article.  1.     Knut Brockow, Medical algorithm: Diagnosis and treatment of radiocontrast media hypersensitivity Allergy 2020 ;75:1278-1280. doi: 10.1111/all.14147. 2.     P. Demoly  N. F. Adkinson  K. Brockow et al. International Consensus on drug allergy, Allergy 2014; 69: 420-437.  https://doi.org/10.1111/all.123503.     Mona Al-Ahmada and Tito Rodriguez Bouzab ,Successful desensitization to radiocontrast media in two high-risk cardiac patients. Ann Saudi Med. 2017; 37(4): 333–335. doi: 10.5144/0256-4947.2017.333 4.     Saurav Uppal , Anthony E DeCicco , Anselma Intini  et al. Rapid Desensitization to Overcome Contrast Allergy Prior to Urgent Coronary Angiography. Int Heart J  2018; 30; 59(3):622-625. doi: 10.1536/ihj.17-395.

To the Editor,We appreciate the interest and comments of Soriano and Ancochea1 regarding our papers 2. Further to the suggestion that “it would be of interest to repeat their statistics conducted during the first wave of COVID-19, again with the current estimates during the ongoing second wave, or later ones”, we would like to emphasize that our geographical observation was a type of anecdotal evidence that contributed to formulating a hypothesis. In a previous paper, we found that after adjusting for potentially relevant country-level confounders, there was a negative ecological association between COVID-19 mortality and the consumption of cabbage and cucumber in European countries 3. In this study, we acknowledged that “As in any ecological study, any inference from the observed association should be made at the country level, as the possibility of ecological fallacy precludes inferences at the individual level; and that further testing in properly designed individual studies would be of interest”. Indeed, what would be useful is testing the hypothesis in robust observational studies and/or clinical trials.Regarding our observation that COVID-19 could be considered as a disease of the Anthropocene 4 , other authors have recently provided a more complete description of the links between the disruption of the natural ecosystems that characterize the Anthropocene and the occurrence of zoonosis 5 6.1. Soriano J and Ancochea J. Saved by cabbage, killed by cabbage, and COVID-19. Allergy 2020; in press.2. Bousquet J, Anto JM, Czarlewski W, et al. Cabbage and fermented vegetables: from death rate heterogeneity in countries to candidates for mitigation strategies of severe COVID-19. Allergy 2020. DOI: 10.1111/all.14549.3. Fonseca S, Rivas I, Romaguera D, et al. Association between consumption of vegetables and COVID-19 mortality at a country level in Europe. MedRix 2020; 10.1101/2020.07.17.201558464. O’Callaghan C and Anto J. COVID-19: The Disease of the Anthropocene.Env Res 2020; 187: 109683.doi: 109610.101016/j.envres.102020.109683. Epub 102020 May 109615.5. Morens DM and Fauci AS. Emerging Pandemic Diseases: How We Got to COVID-19. Cell 2020; 182: 1077-1092. 2020/08/28. DOI: 10.1016/j.cell.2020.08.021.6. Roche B, Garchitorena A, Guegan JF, et al. Was the COVID-19 pandemic avoidable? A call for a ”solution-oriented” approach in pathogen evolutionary ecology to prevent future outbreaks. Ecol Lett 2020 2020/09/02. DOI: 10.1111/ele.13586.JM AntoISGlobAL, Centre for Research in Environmental Epidemiology (CREAL), Barcelona, Spain. IMIM (Hospital del Mar Research Institute), Barcelona, Spain. Universitat Pompeu Fabra (UPF), Barcelona, Spain. CIBER Epidemiología y Salud Pública (CIBERESP), Barcelona, Spain.J BousquetCharité, Universitätsmedizin Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Comprehensive Allergy Center, Department of Dermatology and Allergy, Berlin, Germany. MACVIA-France, Montpellier, France.

Article Type: News and Views: Groundbreaking Discoveries in ImmunologyTitle: Emollients for the prevention of atopic dermatitisAuthors: Akash Kothari1(https://orcid.org/0000-0003-1980-161X), Arielle Locke2,Thomas Eiwegger1,3,4(https://orcid.org/0000-0002-2914-7829)1Translational Medicine Program, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada2Department of Medicine, National University of Ireland, Galway, Ireland3Division of Immunology and Allergy, Food Allergy and Anaphylaxis Program, The Department of Pediatrics, Hospital for Sick Children, Toronto, Ontario, Canada4Department of Immunology, Faculty of Medicine, University of Toronto, Toronto, Ontario, CanadaCorrespondence to: Thomas Eiwegger, MD, Division of Immunology and Allergy, Food Allergy and Anaphylaxis Program, The Department of Paediatrics, Hospital for Sick Children, 555 University Ave, Toronto, Canada, E-mail: [email protected], Tel.: +1 416-813-7654 ext. 1862Conflicts of Interest: AK and AL have nothing to disclose. TE reports to act as local PI for company sponsored trials by DBV and sub-investigator for Regeneron, holds grants from Innovation Fund Denmark, CIHR outside the submitted work. He is Co-Investigator or scientific lead in three investigator initiated oral immunotherapy trials supported by the Food Allergy and Anaphylaxis Program SickKids and serves as associate editor for Allergy. He/his lab received unconditional/in-kind contributions from Macro Array Diagnostics and an unrestricted grant from ALK. He holds advisory board roles for ALK.Financial support: This work was supported by The Hospital for Sick Children, The Food Allergy and Anaphylaxis Program at The Hospital for Sick Children, and The Dr Lorus J And Dr Margery J Milne Scholarship from Victoria University at the University of Toronto.Statement of Author Contribution: All authors critically reviewed the original articles (references 6 and 7) and wrote the News & Views: Groundbreaking discoveries in Immunology article. All authors contributed, revised, edited, and approved the final version of the manuscript as submitted and agreed to be accountable for all aspects of the work.Keywords: emollient, atopic eczema, infancyAbbreviations : food allergy, FA; filaggrin gene, FLG; atopic dermatitis, AD; transepidermal water loss, TEWL; Barrier Enhancement for Eczema Prevention, BEEP; Preventing Atopic Dermatitis and Allergies, PreventADALL

Sustained milk consumption after 2 years post-Milk Epicutaneous therapy for Eosinophilic EsophagitisJonathan M. Spergel, MD, PhD1,2; Amanda B. Muir, MD2,3; Chris A. Liacouras, MD2,3;Deirdre Burke1, CRA; Megan O. Lewis, MSN, RN, CPNP1; Terri Brown-Whitehorn, MD1,2, Antonella Cianferoni, MD, PhD1,21Division of Allergy and Immunology, The Children’s Hospital of Philadelphia, PA, USA, 2Department of Pediatrics, The Perelman School of Medicine at University of Pennsylvania, Philadelphia, PA, USA, 3Division of Gastroenterology, Hepatology, and Nutrition, The Children’s Hospital of Philadelphia, PA, USAAuthor contribution:JMS-study design, writing manuscript, interpreting data; ABM-writing manuscript, study visits, data interpretation; CAL-writing manuscript, study design, interpreting data; DB-writing manuscript, regulatory coordinator, coordination and collection of data, study visits; MOL-writing manuscript, collection of data, study visits; TBW-writing manuscript, collection of data and study visits, AC-interpreting data, collection of data, study visits, regulatory itemsTo the Editor:Eosinophilic Esophagitis (EoE) is an allergic disease of the esophagus without any curative therapy. Typical symptoms of EoE are feeding difficulties, vomiting, abdominal pain and dysphagia and vary by age, with a diagnosis confirmed on esophageal biopsy with> 15 eosinophils/high power field (eos/hpf).1 The two treatment options for pediatric EoE2 are: 1) topical swallowed steroids, which is effective in inducing EoE remission in 50-90% of patients, depending on the dose, formulation and medication used; 2) dietary elimination of the causative allergen/s which is effective in 50-70% of patients with selective food elimination, and 95% with elemental diets3. Cow’s milk (CM) is the most common food allergen causing disease in up to 65% of patients.4 When either treatment is discontinued, inflammation and symptoms recur.3Epicutaneous immunotherapy (EPIT) is an investigational immunotherapy using low dose allergen exposure through the skin to induce desensitization. In the randomized controlled clinical trial,Study of Efficacy and Safety of Viaskin® Milk for CM-induced EoE (SMILEE Study) , 20 pediatric participants with CM-induced EoE were randomized to receive EPIT with Viaskin® Milk (n=15) or placebo (n=5)(details in appendix). After CM-induced EoE was confirmed, EPIT therapy was applied daily for 9 months during a CM-free period, followed by CM-containing diet for 2 months (Figure 1). At 11 months, subjects completed an upper endoscopy with biopsy to evaluate tissue eosinophilia as the primary endpoint. In the pre-defined per-protocol population (7 patients-Viaskin® Milk, 2 patients- placebo), Viaskin® Milk treated subjects had a lower number of eosinophils/high power field (eos/hpf) on biopsy (25.57 ± 31.19) compared to placebo (95 ± 63.64). After the blinded phase, 19 subjects were eligible to enroll in the open-label extension (additional 11 months of therapy) and had repeat endoscopy and biopsy. At the end of the open-label phase, 6/19 subjects had < 6 eos/hpf (32% response rate); 3/19 subjects had 7-14 eos/hpf for total response rate of 47%.5As part of routine clinical care, we continue to follow all 19 subjects who completed the open-label extension (currently 2 years after the end of Viaskin® Milk therapy) to understand whether CM continued in their diet without symptoms. Four of 5 subjects who had <6 eos/hpf after milk introduction were able to continue with approximately 2 servings of CM/day without any symptoms (Table 1). One of these patients had a clinically indicated endoscopy and biopsy that had 0 eosinophils. Two subjects, who had 6-14 eos/hpf during the study, continued to tolerate CM, including one subject who continued to have 6-14 eos/hpf on repeat endoscopy. In addition, 4 subjects who had significant symptoms ingesting CM and had > 15 eos/hpf during the initial SMILEE study were able to add CM back into their diet without having symptoms, as either baked CM (n=2) or regular CM with concomitant swallowed steroids therapy (n=2).The follow-up of this pilot study for the use of EPIT for milk-induced EoE suggests that the treatment effect can persist for 2 years after stopping therapy; six out of 7 patients in the responder and partial responder groups remain completely symptom-free while consuming an average of 2 servings/day of CM. In contrast to the current therapies of diet elimination or swallowed topical steroids where symptoms return when therapy is stopped, EPIT has demonstrated a persistent effectiveness. These findings align with EPIT’s proposed mechanism of action, by directly targeting and reprogramming the immune response to allergen.3 EPIT may induce true tolerance, as is observed in murine models, where Foxp3(+) CD25(+) CD4(+) T regulatory cells are induced and can transfer tolerance.6 Further longer-term studies are needed to examine this possibility and confirm these unique findings.Reference:1. Spergel JM, Dellon ES, Liacouras CA, et al. Summary of the updated international consensus diagnostic criteria for eosinophilic esophagitis: AGREE conference. Ann Allergy Asthma Immunol.2018;121:281-284.2. Spergel JM, Brown-Whitehorn TA, Muir A, Liacouras CA. Medical algorithm: Diagnosis and treatment of eosinophilic esophagitis in children. Allergy. 2020;75:1522-1524.3. Nhu QM, Aceves SS. Medical and dietary management of eosinophilic esophagitis. Ann Allergy Asthma Immunol.2018;121:156-161.4. Kagalwalla AF, Amsden K, Shah A, et al. Cow’s milk elimination: a novel dietary approach to treat eosinophilic esophagitis.J Pediatr Gastroenterol Nutr. 2012;55:711-716.5. Spergel JM, Elci OU, Muir AB, et al. Efficacy of Epicutaneous Immunotherapy in Children With Milk-Induced Eosinophilic Esophagitis. Clin Gastroenterol Hepatol. 2020;18:328-336 e327.6. Dioszeghy V, Mondoulet L, Dhelft V, et al. The regulatory T cells induction by epicutaneous immunotherapy is sustained and mediates long-term protection from eosinophilic disorders in peanut-sensitized mice. Clin Exp Allergy. 2014;44:867-881.Sincerely,Jonathan M. Spergel, MD, PhD1,2; Amanda B. Muir, MD2,3; Chris A. Liacouras, MD2,3;Deirdre Burke1, CRA; Megan O. Lewis, MSN, RN, CPNP1; Terri Brown-Whitehorn, MD1,2, Antonella Cianferoni, MD, PhD1,21Division of Allergy and Immunology, The Children’s Hospital of Philadelphia, PA, USA, 2Department of Pediatrics, The Perelman School of Medicine at University of Pennsylvania, Philadelphia, PA, USA, 3Division of Gastroenterology, Hepatology, and Nutrition, The Children’s Hospital of Philadelphia, PA, USAFunding Sources: The Children’s Hospital of Philadelphia Eosinophilic Esophagitis Family FundAcknowledgements: JMS, CAL, TBW and MOL are consultants for DBV Technology.Correspondence: Jonathan M. Spergel MD, PhDThe Children’s Hospital of PhiladelphiaDivision of Allergy and ImmunologyWood Bldg 3352D3401 Civic Center Blvd,Philadelphia, PA 19104Email: [email protected]: 1-215-590-2549Table 1

To the Editor, Immediate hypersensitivity reactions are related to mast cell and/or basophil activation. The mediators released such as tryptase and histamine are involved in clinical symptoms and are key parameters that contribute to diagnosis. Serum tryptase concentrations peak between 30 minutes and 4 hours following the reaction. Tryptase release is considered a robust marker of mast cell degranulation but is not informative in mild reactions.1 Histamine is released at the early beginning of the reaction but has a short half-life. The concentration value can be altered by pre-analytic conditions.2 Anaphylactic reactions can occur during night and week-end when laboratories can’t take charge of samples. To our knowledge, no thorough study of the stability in whole blood of these markers in anaphylaxis has been published. The aim of our study was to evaluate the impact of whole blood sample storage conditions (temperature and delay before centrifugation and plasma collection) on the reliability of tryptase and histamine measurements.Blood samples from 14 patients suspected of anaphylactic reactions (grade 2 to 4 of the Ring and Messmer scale)3 and from 10 volunteers were collected on EDTA after signed informed consent (CCPPRB Caen Basse-Normandie protocol 2004-32). The description of the patient anaphylactic episodes appears in Table 1.When received in the lab, an aliquot of whole blood was processed for diagnostic (reference measurement) and the remaining was divided in aliquots stored at room temperature (RT) or at +4°C for 24, 72 hours or 7 days (patients) or 2, 6, 24 or 72 hours for controls before centrifugation and plasma collection.Total tryptase concentrations were measured by an automated fluoroimmunoassay (ThermoFisher, Phadia SAS, ). Increased tryptase is defined as ≥ 1.2 x basal value + 2 µg.L-1.4 In our hands, tryptase uncertainties of measurement for low and high concentrations (9 µg.L-1 and 38.2 µg.L-1) are 17% and 16% respectively, in accordance with published results.5 Plasma histamine concentrations were measured by a radioimmunoassay (Beckman Coulter, Immunotech, France). Increased values defined by the manufacturer are >10 nmol.L-1, in accordance with published data.6 In our hands, histamine uncertainties of measurement for low and moderate concentrations (4.7 nmol.L-1 and 12.9 nmol.L-1) were 22% and 25%, respectively.The differences between the concentrations measured before and after storage were compared by paired two-tailed t-tests using SAS software. Results were considered significant for p < 0.05.As shown in Figure 1A, storage conditions did not modify tryptase concentrations (linear regression: slope=1.079, R²=0.9675). Tryptase concentrations appeared stable in whole blood left at +4°C for 7 days or 72 hours at RT.Histamine concentrations in patient samples were not modified during 72h at +4°C (Figure 1B) or at RT (Figure 1C). In the control group at RT, histamine concentrations were significantly increased at 6 hours (p=0.005) although moderately increased and staying within the limits of uncertainty measurement and never reaching the positivity threshold (Figure 1E). After 24 hours at RT a false positivity was observed for 8 of 10 samples (p<0.0001) (Figure 1E). At +4°C, histamine concentrations were significantly increased after 24 hours (p<0.0001) but remained in the limits of uncertainty measurement and under the threshold of positivity (Figure 1D). After 72h at +4°C, histamine concentrations exceeded the limits of uncertainty measurement and the positivity threshold for 4 samples of 10 (Figure 1D).Tryptase and histamine measurements are recommended to prove degranulation in anaphylaxis.7 Anaphylactic reactions occur unexpectedly. It is thus important to master sample shipment and processing before mediator measurement.The knowledge of possible artifacts modifying the measured values is necessary for the biochemist to address accreditation criteria of pre-analytic requirements (ISO 15189 standard) and for the physician to rely on trustable diagnostic data.Tryptase stability in whole blood had not been described. Our data has shown no impact on results after 72h at RT or 7 days at +4°C. Tryptase stability in plasma or serum has been evaluated by the manufacturer who ensured stability for 48h at RT or 5 days at +2°C to +8°C (Thermofisher).8 Thus, measured values of tryptase appear highly reliable.Histamine stability in whole blood had only been evaluated in controls and false positive results may be attributed to passive release from basophil during prolonged storage.2 We observed no impact for patient blood samples after 72h at +4°C or at RT. In contrast, false positive results were observed in controls after storage at RT during 24h or at +4°C during 72h. Histamine is known to be stable in the plasma obtained after centrifugation up to 4 days at RT for patients and controls.9According to these results, we suggest that whole blood samples can be stored at +4°C up to 72h for histamine and 7 days for tryptase when the laboratory is not available immediately. In any case, the biochemist must accept all these unrenewable samples. It is his role to take into account the pre-analytical conditions to interpret the results and provide helpful information to the physician.Keywords : pre-analytic; tryptase; histamine; whole blood.

Brain activation after nasal histamine provocation in house dust mite allergic rhinitis patientsCallebaut I1, Steelant B1, Backaert W1, Peeters R2-3, Sunaert S2-3, Van Oudenhove L4-5*, Hellings PW1*1Allergy and Clinical Immunology Research Group, Department of Microbiology, Immunology and Transplantation, KU Leuven, Leuven, Belgium2Department of Imaging & Pathology, KU Leuven, Leuven, Belgium3Department of Radiology, University Hospitals Leuven, Leuven, Belgium4Laboratory for Brain-Gut Axis Studies (LaBGAS), Translational Research Center for Gastrointestinal Disorders (TARGID), Department of Chronic Diseases, Metabolism, and Ageing (CHROMETA), University of Leuven, Belgium5Cognitive and Affective Neuroscience Laboratory (CANlab), Center for Cognitive Neuroscience, Department of Psychological and Brain Sciences, Dartmouth College, Hanover, NH, USA*Joined senior authorshipTo the editor . The nasal mucosa is armed with a complex nervous system of sensory, sympathetic and parasympathetic nerves, allowing swift defensive responses to physical and chemical stimuli. In allergic rhinitis (AR) patients, nasal allergen deposition leads to mast cell activation with release of allergic mediators such as histamine. Apart from its direct effects on the surrounding tissue, histamine also activates sensory nerve endings giving rise to symptoms like sneezing, rhinorrhoea, and/or congestion(1). Activated nasal sensory nerves transmit action potentials to their cell bodies in the trigeminal ganglion and further to the midbrain where secondary synapses lead to the generation of central reflex signals. Despite activation of neural pathways in AR(2), it is not known which particular regions in the brain are activated by different nasal stimuli. Clinical studies using Positron Emission Tomography scans indicate that there is no isolated itch center in the brain but that different cortical centers are involved in the processing of itch(3, 4). Activation of the anterior cingulate cortex (ACC), the supplementary motor area (SMA), and the inferior paretial lobe partly explains the connection between itching and the related reflex of scratching(4). Using functional magnetic resonance imaging (fMRI), the activation of the superior temporal gyrus, insula and nucleus caudate following painful intranasal trigeminal stimulation has been shown(5). When asthmatic patients are challenged with metacholine or allergens, activity in ACC and insula was associated with markers of bronchial inflammation and obstruction(6).To fill the abovementioned knowledge gap, a prospective, single-blind, cross-over study was designed to investigate brain responses to nasal histamine provocation in healthy volunteers and AR patients.Eight house dust mite (HDM) AR patients and 7 non-allergic healthy controls (HC) were recruited at the outpatient clinic for Otorhinolaryngology of University Hospitals Leuven. HDM allergy was confirmed by a skin prick test. Relevant nasal anatomic abnormalities or rhinosinusitis were ruled out by nasal endoscopy. Non-allergic HC showed a negative skin prick test for all the tested allergens, showed no nasal symptoms and had normal nasal endoscopy. Patients of <18 and >50 years of age, having used nasal or oral steroid treatment <6weeks prior to the study or nasal or oral antihistamine treatment <4weeks prior to the study were excluded, as well as those with past or ongoing immunotherapy for HDM, asthma, smoking and clinical signs of rhinosinusitis or anatomic nasal deformities. Informed consent was signed by all participants. The study was approved by the local medical ethics committee of the University Hospitals Leuven (B322201215751).All HC and AR patients underwent a nasal provocation by means of a canulla placed under the nose with either nebulized sham solution (saline) or with histamine for 5 minutes while in supine position in the MR scanner on 2 separate days with a minimum of 1 week in between, and in a single-blinded and random order. An aerosol of 10 ml histamine HCl (16 mg/ml) or 10 ml saline was delivered via the canulla by means of air (8 bar) after 10 minutes of baseline scanning in a pharmacological (ph)MRI design. This concentration of histamine was chosen as optimal dose after a pilot study in 3 HCs, 1 birch and grass pollen AR patient and 1 HDM AR patient where the dose of histamine resulted in a reduction of 20% in the Peak Nasal Inspiratory Flow (PNIF). Moreover, patients did not had the urge to sneeze at this concentration, as was the case for the dose of 32 mg/ml.PNIF values were used for measuring nasal flow at baseline and after the nasal provocation at the end of the phMRI scan, as recommended(7). The best value out of three consecutive measurements with a variability of <10% was recorded. Changes in PNIF from baseline to post-provocation were compared between conditons (histamine & saline) as well as between groups (patients & controls) using marginal linear mixed models.phMRI data were preprocessed and analyzed as described previously(8, 9). The effect of interest for the present study was the group (patient versus controls)-by-substance (histamine versus saline)-by-time interaction effect, comparing the time-course of the brain response to histamine vs saline provocation between AR patients and controls. A whole-brain voxel-wise FWE-corrected threshold of p<0.05 was used combined with an extent threshold of k=10 voxels (corresponding to pFWE<0.001 at cluster level).In total, 8 HDM AR patients (5 females and 3 males) and 7 HC (5 females and 2 males) were recruited with a mean age of 22.5 ± 0.72 and 23.8 ± 1.11 years respectively. One female HDM AR and two female HC were excluded due to excessive head movement during MR scanning.After nasal provocation with saline, no significant decrease in PNIF was found compared to baseline in both groups (AR: 135 ± 61.82 l/min vs 137.5 ± 44.88 l/min, p=0.74; HC: 120 ± 36.74 vs 129 ± 31.30, p=0.46). Nasal provocation with histamine induced a significant decrease in PNIF in both HDM AR patients (158.8 ± 71.55 l/min vs 112.5 ± 83.67, p=0.0053) as well as in the HC (134.2 ± 27.64 l/min vs 85.83 ± 40.55, p=0.002).The analysis on PNIF values showed a significant condition-by-time (pre- to post-provocation) interaction effect (F(1,11)=28.8, p=0.0002), driven by a significant decrease in PNIF after histamine (-47.30±8.87, pHolm=0.0004), but not after saline (-5.81±5.96, pHolm=0.35) in the entire sample. No significant group-by-condition-by-time interaction effect was found (F(1,11)=0.09, p=0.78) indicating that the decrease from baseline after histamine compared to saline did not differ between patients and controls, with a significant decrease from baseline after histamine but not saline in both groups (p=0.002 and p=0.015, respectively).Brain regions showing a differential response to histamine versus saline in AR patients versus HCs included bilateral mid-/posterior insula, right anterior insula, bilateral postcentral/superior temporal gyrus/rolandic operculum (including secondary somatosensory cortex), bilateral putamen, left cerebellum (crus 1 & 2), right mid-occipital gyrus, bilateral medial orbital gyrus/gyrus rectus, and right middle/superior frontal gyrus (ventrolateral prefrontal cortex) (Table 1,Figure 1). Most of these differential responses were due to a stronger activation in controls vs AR patients, except for the right anterior insula, right middle occipital gyrus, right middle/superior frontal gyrus, and left cerebellum, where a stronger activation was observed in AR patients.

Title: Real-life study in non-atopic severe asthma patients achieving disease control by omalizumab treatmentTo the Editor,Severe asthma is defined as asthma requiring treatment with guidelines-suggested medications for Global Initiative for Asthma (GINA) steps 4 or 5 or systemic corticosteroids for ≥50% of the previous year to prevent it from becoming ‘uncontrolled’ or which remains ‘uncontrolled’ despite this therapy.1 Up to 34%–50% of severe asthmatic patients have non-atopic (also called non-allergic) asthma. 2 A significant proportion of these patients have severe uncontrolled asthma, which requires high doses of inhaled corticosteroids (ICS) or even oral corticosteroids (OCS).2 Until the advent of biologics, treatment options in these patients have been very limited. For many years, both the pathogenesis knowledge and the results of clinical trials supported the view that anti-IgE treatment is specifically effective in allergic asthma. Interestingly, recent molecular and clinical evidence suggests that anti-IgE treatment might also be effective in patients with non-allergic asthma.2 Omalizumab (Xolair®) is an anti-IgE monoclonal antibody that selectively binds to human IgE and prevents the binding of IgE to its receptors. Although omalizumab is indicated in Europe in patients with severe persistent allergic asthma, several case reports and short series have provided data on the value of omalizumab in patients with non-atopic asthma.3,4The observational, multicenter, retrospective, real-life FENOMA study specifically evaluated patients who achieved full asthma control after one year of treatment with omalizumab.5 The study included 345 patients, 80 (23.2%) of whom had non-atopic asthma. The present post-hoc sub-analysis aims to describe the clinical improvement of patients with non-atopic asthma. Socio-demographic and asthma-related characteristics were collected at baseline. Outcomes analyzed at baseline and after one year of treatment were those included in the definition of asthma control by GEMA guidelines.5 Medical records were reviewed between February 2015 and June 2016. For statistical comparisons, the 2-sided Wilcoxon signed-rank test was used. A P-value of <0.05 was considered to be statistically significant. All analyses were performed with the SAS statistical package (version 9.4; SAS Institute, Cary, NC).The primary outcome of this post-hoc sub-analysis was to describe the baseline characteristics and clinical improvement of non-atopic asthma patients who achieved full disease control after one-year of treatment with omalizumab through i) frequency of daytime symptoms, ii) changes in use of ICS or OCS iii) need for rescue therapy, iv) pulmonary function (forced expiratory volume in 1 second [FEV1]), v) number of non-severe exacerbations and vi) use of healthcare resources, i.e. unplanned visits to primary care or specialists and the number of days of school or workplace absenteeism due to asthma worsening. Non-severe asthma exacerbations were defined as those that did not require OCS, emergency assistance or hospitalization. Secondary outcomes include an assessment of the percentage of eosinophil blood count and exhaled nitric oxide fraction (FeNO) before and after treatment.Demographic, clinical characteristics and asthma history (before starting treatment with omalizumab) are shown in Table 1 . Mean (SD) age of patients was 58.7 (12.2) years and 65% were female. Almost all patients had daytime symptoms, 92% of patients needed rescue medication, and the mean (SD) initial dose of omalizumab was 338.7 (153.1) mg.After one year of treatment with omalizumab 50.0% (n=40) of patients had no daytime symptoms, while 37.5% (n=30) and 12.5% (n=10) had symptoms 1 and 2 days per week, respectively. Forty-one (51.2%) of the 54 patients who were receiving OCS at entry, stopped treatment (P<0.0001). Of those continuing on OCS, the average reduction of the daily dose was not statistically significant (P=0.2132). More than half of patients (53.7%, n=43) needed no rescue medication. Median FEV1 increase was 15% and there was a reduction in the number of non-severe asthma exacerbations. After one year of treatment with omalizumab, a great reduction in unplanned visits and absenteeism from school or workplace (P<0.0001; Table 2 ) was observed.Of note, the effectiveness of omalizumab was previously assessed in a Spanish multicenter registry, which evaluated 29 non-atopic severe asthma patients over 2 years.6 However, our series is the most extensive study in patients with non-atopic asthma published to date in Spain, and provides data on full disease control. There have been several potential suggestions to explain the effectiveness of omalizumab in non-atopic patients.7 In a proof-of-concept study in non-atopic asthma patients, treatment with omalizumab resulted – as per in atopic patients – in a significant reduction of high-affinity IgE receptor (FcεRI) expression on blood basophils and plasmacytoid dendritic cells (pDC2), which hampered IgE binding and the subsequent production on proinflammatory mediators.8 Additionally, omalizumab treatment was associated with an increase in FEV1 with a positive trend in some relevant clinical endpoints, such as asthma exacerbations.8 In another proof-of-concept trial, omalizumab therapy (but not placebo) reduced IgE expression and IgE sensitization of target cells within the bronchial mucosa, and increased FEV1 versus baseline despite withdrawal of conventional therapy.9 Interestingly, it has been hypothesized that patients labelled as ‘non-allergic’ might in fact have a localized allergy to an unrecognized allergen, with elevated concentrations of allergen-specific IgE antibodies in the airways.7Our study has several limitations. Its single-arm retrospective nature relies on the accuracy and completeness of the information entered into the clinical records. This has especially affected predictors of response such as FeNO and the level of eosinophils, which were not routinely assessed in the clinical practice at the time of the study. The benefits of omalizumab presented here are those observed in the population of non-atopic patients who achieved disease control after one year of treatment with omalizumab. It is unknown how many other patients classified as non-atopic in the clinical practice did not benefit from this treatment.In summary, in the population of patients with non-atopic severe asthma who achieved full disease control after one year of treatment with omalizumab, the clinical and pulmonary benefits were remarkable and similar to those described for atopic patients. A reduction in the use of healthcare resources was also documented. Large randomized controlled trials are warranted to confirm the value of omalizumab in this population of patients.

A document by Shen Zhang. Click on the document to view its contents.

The role of eosinophils in allergic inflammation is well recognized. In homeostasis these cells are found in multiple healthy tissues including the lung parenchyma, but the function of these resident eosinophils is unknown. Circulating eosinophils are easily quantifiable and have been used to define “eosinophilic phenotype”, and to select patients who are likely to respond to anti-eosinophil and anti-Th2—directed therapies. However, presence of eosinophils in circulation may not necessarily indicate that the eosinophils are key effector cells for an airway disease such as asthma and this may be reason for not all patients responding well to anti-IL5 therapies despite normalization of blood eosinophils. This pro-con commentary examines the role of enumerating circulating vs luminal (sputum) eosinophils (and their activation status) not only to initiate therapies with monoclonal antibodies, but to monitor their clinical response while on therapy.

To the Editor.The beneficial effects of Allergen Specific Immunotherapy (AIT) relies on the induction of allergen-specific Regulatory T-cells (Tregs) (1). Tregs, a subpopulation of CD4+CD25+T-cells expressing the specific transcription factor Foxp3, are not functionally homogeneous and their detection is complex and uncertain due to FoxP3 intracellular localization. Furthermore, FoxP3+ Tregs might become unstable and halt the production of their functional suppressive cytokines in inflammatory conditions (2) (1). In its place, the surface antigen CD127, whose expression inversely correlates with FoxP3, conveniently identifies Tregs as CD4+CD25+CD127negcells (3) (2), so surmounting the problems of FoxP3 stability and intracellular detection. Tregs also constitutively express the inhibitory antigen CD39, enhanced in highly suppressive memory Tregs (4) (3). Furthermore, HLA-DR expression is a monitor of Treg differentiation status and identifies a functionally and greatly suppressive population (1,5) (1,5). Lack of CD45RA characterizes memory T cells enabled to survive for long periods, even in absence of specific antigen, showing increased activity upon re-exposure and able to induce apoptosis in target cells (6) (3). CD4+CD25highCD39+CD127negcells are subtyped as Resting (CD45RA+/HLA-DRneg: rTreg), Activated (CD45RAnegHLA-DRneg: aTreg) and Effector (CD45RAnegHLA-DRlow/high: eTreg) Tregs (6) (6). This latter subtype includes terminally differentiated Tregs, the most highly suppressive (5) (7) (Supplementary Figure 1). They are different from secreting or type III Tregs expressing CD127 that represent a short-lived terminally differentiated population (5,6,8) . In order verify possible correlations between specific subsets of Treg and the effectiveness of AIT, we applied this analytical approach to study Treg profile in adolescents suffering from mite allergic rhinitis, pre and 12 months post Sublingual Immunotherapy (SLIT) with mite monomeric allergoid, an acid-resistant allergen known to elicit early T reg-activation (7,8). The study was approved by the Ethical Committee of University “G. d’Annunzio”, Chieti-Pescara. All patients and parents signed a written informed consent after having been informed about the procedures of the study.Twenty patients diagnosed with mite-allergic persistent rhinitis with or without asthma were enrolled. Allergic rhinitis (AR) was graded according to ARIA guidelines in 1) intermittent mild, 2) intermittent moderate/severe, 3) persistent mild and 4) persistent moderate/severe. At the enrollment, each patient marked in a 100 mm visual analogic scale (VAS) the level of its health status related to allergy with 0 the best status and 100 the worst.All patients were treated by SLIT with mite monomeric allergoid (LAIS - Lofarma, Milan, Italy) at 1000 UA four times/week every other day, for 12-months. No adverse local and systemic reactions were detected. The effectiveness of SLIT was established comparing VAS, ARIA grading and ACT questionnaire performed after 12-months of treatment with their basal values. Two blood samples were drawn pre/post SLIT to be analyzed for Regulatory T-cells. Clinical and demographic details of the studied population, analytical methods, statistical approach and the outline of the study are detailed in the online supplementary material .Rhinitis scores VAS and ARIA significantly decreased after SLIT (Table 1), with the same statistical significance (Wilcoxon z -3.7236; p = 0.0002). Improvement was evidenced also in the subgroup of asthmatic patients (n=7) since ACT scores significantly increased from the baseline value of 18 (16-19) up to 24 (20-25) after 12 months of treatment (the low number of patients does not allow application of efficient statistics).Tregs were analyzed as frequency of total Treg cells and their three subsets, namely Resting (rTregs), Activated (aTregs) and Effector (eTregs), within the parental population of CD4+cells. Total Tregs did not change significantly; rTreg significantly decreased (Wilcoxon z-3.6214, p<0.0003), while, the abundance of aTregs and eTregs significantly incremented (Wilcoxon z-2.9011, p<0.05 and z-3.077, p=0.002, respectively) (Table 1). A significant negative correlation has been observed between the decrease in rTreg and the increase in aTreg (Spearman’s ρ-0.69391, p<0.02) and increase in eTreg cells (Spearman’s ρ-0.56845, p<0.02) (Figure 3 in supplementary material).HLA-DR resulted significantly up-regulated in all Tregs from 4.93±3.1 to 6.92±5.1 MFI (Wilcoxon z-4.2026, p <0.00001). HLA-DR increased on aTregs from 3.4±3.03 to 4.91±3.2 MFI (Wilcoxon z-3.2479, p=0.001) and on eTregs from 1.54±0.66 to 2.0±1.45 MFI (Wilcoxon z-2.9664, p=0.005). CD39 was found differently expressed in the three subsets of Tregs at baseline, with Resting<Activated<Effector. After 12 months of SLIT, CD39 surface expression was found significantly increased in all Tregs from 6.9±4 to 8.02±5 MFI (Wilcoxon z-3.1049, p=0.001) (HLA-DR and CD39 changes are reported in Table 1). We found some interesting correlations between laboratory data and clinical parameters. Changes in eTregs significantly correlated with both ARIA (Spearman’s ρ=0.58728, p=0.013) (Figure 1A) and VAS (Spearman’s ρ=0.49172, p=0.044) (Figure 1B) variations after SLIT. While a significant negative correlation was found between rTregs and clinical parameter changes after treatment (Spearman’s ρ-0.48482, p=0.0491). Changes in HLA-DR expression on all Treg cells significantly correlated with variation in VAS pre-/post-SLIT (Spearman’s ρ=0.54104, p= 0.01376) (Figure 1C). No other correlations were found except for the lowest increase (< 8%) of memory Tregs (CD45RAneg) detected in patients with the lowest levels of mite-specific serum IgE (not shown).To our knowledge this is the first report on successful SLIT being associated with re-patterning of the differentiation status of Tregs, with high rates of the most suppressive Treg subtypes: activated and effector, characterized by higher expression of HLA-DR and CD39 both playing inhibitory function in Tregs. Moreover, effective SLIT seems to be associated with the generation of cells lacking CD45RA that characterizes memory T cells with increased activity upon re-exposure to the antigen. Our results suggest that SLIT also induced empowerment of Treg inhibitory function, likely compensating the under-representation of Tregs observed in allergic patients (9) (9). In AR children, there are evidences that Tregs have defect in suppressing IgE production and that they can be incremented by mite SLIT.Next step of our study will be to evidence if such relationship between effective SLIT and Treg re-patterning is present in the first months of SLIT, with a view to profiling Tregs for the early identification of SLIT responders/non-responders by mean of a straightforward and non-invasive blood test.

Medical Algorithm: Early Introduction of Food Allergens in High Risk PopulationsHelen R Fisher,1,2 Gideon Lack,1,2,3 Graham Roberts,4,5,6 Henry T Bahnson,7 George Du Toit.1,2,31Paediatric Allergy Group, Department of Women and Children’s Heath, School of Life Course Sciences, King’s College London, London, United Kingdom2Paediatric Allergy Group, Peter Gorer Department of Immunobiology, School of Immunology and Microbial Sciences, King’s College London, London, United Kingdom.3Children’s Allergy Service, Guy’s and St. Thomas’ NHS Foundation Trust, London, United Kingdom.4The David Hide Asthma and Allergy Research Centre, St Mary’s Hospital, Newport, UK.5NIHR Biomedical Research Centre, University Hospital Southampton NHS Foundation Trust, Southampton, UK.6Faculty of Medicine, Clinical and Experimental Sciences, Human Development in Health Academic Units, University of Southampton, Southampton, UK.7Immune Tolerance Network, Benaroya Research Institute, Seattle, WashingtonCorresponding Authour:Professor George Du ToitPaediatric AllergyBlock B, South WingSt Thomas’ HospitalLondonSE1 7EHTel: 0207 188 9784Email: [email protected] Count: 602Tables: 0Figures: 1Oral Tolerance Induction (OTI) is the only RCT-proven effective intervention for preventing childhood food allergy.(1) OTI to peanut is effective in a general population, with the greatest effect, 81% RRR, noted in the high-risk population.(2) OTI also reduced egg allergy in the general population.(1) Many governmental and allergy societies now recommend introducing peanut in infancy and some suggest other foods, such as well-cooked egg, are also introduced. Choosing which infants should undergo OTI, at what age, to which foods, and under which circumstances is critical for successful OTI prevention in populations where food allergy is a public health concern.Infants with eczema are at increased risk of food allergy but infants from the general population are also at risk and contribute most cases at a population level. Risk of food sensitisation or food allergy increase with age; OTI is most likely to be successful when started in early infancy. Oral tolerance induction from 4 months of age, when completed using standard foods, is safe for nutrition, growth and general child health outcomes (3). Commencing multiple food OTI at 4 months of age, has no detrimental effect on established breastfeeding.(4) All children should adopt a diverse weaning diet, including allergenic foods such as well-cooked egg and peanut, as soon as weaning commences. High risk children should not delay weaning but start weaning and actively include peanut and well-cooked egg, as soon as developmentally ready; usually at about 4 months of age (Fig 1).A 2g/week dosing regime of peanut and well-cooked egg in early infancy is more effective in inducing oral tolerance than later introduction.(5) A lower dosing regime has not been shown to be effective in preventing allergy but, importantly, does not increase allergy risk above that of children who introduce allergenic foods in later infancy.(4) There are limited data regarding the efficacy of OTI to other allergenic foods, or the dose required.(1) All infants should aim to consume about 2g of peanut protein and well-cooked egg per week; parents of high-risk infants should give these amounts more diligently. Given the benefit observed for peanut and egg, it is reasonable for all weaning infants to additionally incorporate 2g of other common and nutritious food allergens; cow’s milk (e.g. as yoghurt), wheat, fish and sesame.Whether children should undergo allergy testing and/or have their first feed of peanut under medical supervision is contested. This cautious approach, potentially requiring large numbers of children to access specialist allergy care, must be balanced against the risks of severe allergic reaction, particularly as most allergic reactions occur on first oral exposure. RCTs of OTI using whole foods had no cases of anaphylaxis on first exposure (4, 6) although anaphylaxis has occurred to OTI using pasteurised whole egg powder.(7) Children with no personal food allergy risk factors do not require testing prior to, or medical supervision during, their first consumption of peanut or well-cooked egg. Children with moderate to severe eczema, or with an existing food allergy should undergo allergy testing +/- OFC at a specialist allergy centre(8), if doing so would not cause undue delay to OTI. It is likely that rapid access to allergy services will be further compromised as a consequence of the COVID-19 pandemic. It may however be that access to SpIgE is available through GP or paediatrician which, if ≥0.35KiU/L, will require referral for OFC. If negative (<0.35KiU/L) the food may be introduced at home following precautionary measures for the first feed: child is well; parent is aware of the signs of IgE mediated reaction has, access to medical support if required and age-appropriate form of the food is given incrementally (Figure 1).

A document by Natalija Novak. Click on the document to view its contents.

IgE, the key molecule in atopy has been shown to bind two receptors, FcRI, the high affinity receptor and FcεRII (CD23), mostly found on B cells and that binds IgE with lower affinity. Whereas cross-linking of IgE on FcRI triggers allergic reaction, binding of IgE to CD23 is known to play an important role in both IgE synthesis and presentation. Thus, targeting IgE-immune complexes on B cells has shown to enhance antibody and T cell responses in mice and humans. However, the mechanisms that regulate the targeting of the two receptors and the respective function of the two pathways in inflammation or homeostasis are still matter of debate. Here, we discuss several mechanisms related to IgE and IgE binding to both receptors, as well as the influence of the antigen binding on different immune cells expressing the receptors. One recent paper has shown that free IgE preferentially binds to FcRI whereas IgE immune complexes (IgE-ICs) are preferentially captured by CD23. Binding of IgE-ICs to CD23 on B cells can on one hand regulate serum IgE and prevent effector cell activation and on the other hand facilitate the antigen presentation by delivering antigen to dendritic cells. The data suggest that CD23 play a multifunctional role in regulating the allergic response pathway.

Background. Lymphocyte transformation test (LTT) has been widely used to evaluate non-immediate drug hypersensitivity reactions (NIDHRs). However, the lack of standardisation and the low sensitivity have limited its routine diagnostic use. The drug presentation by dendritic cells (DCs) and the assessment of proliferation on effector cells have shown promising results. Flow-cytometry-based methods can help apply these improvements. We aimed to assess the added value of using drug-primed-DCs and the determination of the proliferative response of different lymphocyte subpopulations in NIDHRs. Methods. Patients with confirmed NIDHR were evaluated by both conventional (C-LTT) and with drug-primed-DCs LTT (dDC-LTT) analysing the proliferative response in T-cells and other effector cell subpopulations by using the fluorescent molecule, carboxyfluorescein diacetate succinimidyl ester. Results. The C-LTT showed a significantly lower sensitivity (33.3%) compared with dDC-LTT (65.2%), which was confirmed analysing each particular clinical entity: SJS-TEN (62.5% vs 87.5%), MPE (14.3% vs 41.7%), and AGEP (33% vs 80%). When including the effector cell subpopulations involved in each clinical entity, CD3++CD4+Th1 cells in SJS-TEN, CD3++CD4+Th1+NK cells in MPE, and CD3++NK cells in AGEP, we could significantly increase the sensitivity of the in vitro test to 100%, 66.6%, and 100%, respectively. With an overall sensitivity of 87% and 85% of specificity in NIDHR. Conclusions. The use of a flow-cytometry-based test, DCs as drug presenting cells, and focussing on effector cell subpopulations for each clinical entity significantly improved the drug-specific proliferative response in NIDHRs with a unique cellular in vitro test.

Background: There is limited information on risk factors for eczema in adults. Recent evidence suggests that air pollution may be associated with increased incidence of eczema in adults. We aimed to assess this possible association. Methods: Ambient air pollution exposures (distance from a major road, nitrogen dioxide [NO2], fine particulate matter with an aerodynamic diameter ≤2.5 µm [PM2.5]) were assessed for the residential address of Tasmanian Longitudinal Health Study participants at ages 43 and 53 years. Eczema incidence (onset after age 43 years), prevalence (at 53 years) and persistence were assessed from surveys, while sensitisation was assessed using skin prick tests. The presence or absence of eczema and sensitisation was classified into four groups: no atopy or eczema, atopy alone, non-atopic eczema, and atopic eczema. Adjusted logistic and multinomial regression models were fitted to estimate associations between ambient air pollution and eczema, and interaction by sex was assessed. Results: Of 3153 participants in both follow ups, 2369 had valid skin prick tests. For males, a 2.3 ppb increase in baseline NO2 was associated with increased risk of prevalent eczema (OR=1.15 [95%CI 0.98-1.36]), both non-atopic (OR=1.39 [1.02-1.90]) and atopic eczema (OR=1.26 [1.00-1.59]). These associations were not seen in females (P for interaction=0.08, <0.01). For both sexes, a 1.6 µg/m3 increase in PM2.5 exposure at follow-up was associated with increased odds of aeroallergen sensitisation (OR=1.15 [1.03-1.30]). Conclusion: Increased exposure to residential ambient air pollutants was associated with an increased risk of eczema, only in males, and aeroallergen sensitisation in both genders.

A document by Asami Fukuda. Click on the document to view its contents.

Nanotechnology is science, engineering, and technology conducted at the nanoscale, which is about 1 to 100 nanometers. It has led to the development of nanomaterials, which behave very differently compared with materials with larger scales and can be applied in a wide range of applications in biomedicine. The physical and chemical properties of materials of such small compounds depend mainly on the size, shape, composition, and functionalisation of the system. Nanoparticles, carbon nanotubes, liposomes, polymers, dendrimers, nanogels, among others, can be nanoengineeried for controlling all parameters, including their functionalisation with ligands, which provide the desired interaction with the immunological system. However, undesired issues related to their toxicity and hypersensitivity responses have impeded more rapid health applications. Through interactions with the immune system, some of these nanostructures show promising applications as vaccines and diagnostics tools. Dendrimeric Antigens, Nanoallergens, and nanoparticles are potential tools for the in vitro diagnosis of allergic reactions. Glycodendrimers, liposomes, polymers, and nanoparticles have shown interesting applications in immunotherapy. There are wide panels of structures accessible, and controlling their physico-chemical properties would allow the obtainment of safer and more efficient compounds for clinical applications goals, either in diagnosis or treatment.

Food allergy is an increasingly common disease worldwide, and is thought to be driven by an uncontrolled type 2 immune response. Current knowledge about the underlying mechanisms that initiate and promote an inappropriate immune response to dietary allergens is limited. Sensitization through the skin in early life is considered to be a key event. Food allergy results from a dysregulated type 2 response to food allergens, characterized by enhanced levels of IgE, IL-4, IL-5 and IL-13 with infiltration of mast cells, eosinophils and basophils during acute reactions. Recent data implies a possible role of innate lymphoid cells (ILCs) in driving food allergy. ILCs represent a group of lymphocytes that lack specific, recombined antigen receptors. They contribute to immune responses not only through the release of cytokines and other mediators, but also by responding to cytokines produced by activated cells in their local microenvironment. Due to their localization at barrier surfaces of the airways, gut and skin, ILCs form a link between the innate and adaptive immunity. This review summarizes recent evidences on how skin and gastrointestinal mucosal immune system contribute to both homeostasis and the development of food allergy, as well as the involvement of ILCs towards inflammatory processes and regulatory mechanisms.